CN102448500A - Conjugation methods - Google Patents

Conjugation methods Download PDF

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CN102448500A
CN102448500A CN2010800243253A CN201080024325A CN102448500A CN 102448500 A CN102448500 A CN 102448500A CN 2010800243253 A CN2010800243253 A CN 2010800243253A CN 201080024325 A CN201080024325 A CN 201080024325A CN 102448500 A CN102448500 A CN 102448500A
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conjugates
antibody
connector
described method
mixture
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B·A·克洛格
R·辛
R·V·J·查理
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Immunogen Inc
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Immunogen Inc
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Priority to CN201510338095.2A priority Critical patent/CN104984360A/en
Publication of CN102448500A publication Critical patent/CN102448500A/en
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Abstract

This invention describes a method of conjugating a cell binding agent such as an antibody with an effector group (e.g., a cytotoxic agent) or a reporter group (e.g., a radionuclide), whereby the reporter or effector group is first reacted with a bifunctional linker and the mixture is then used without purification for the conjugation reaction with the cell binding agent. The method described in this invention is advantageous for preparation of stably-linked conjugates of cell binding agents, such as antibodies with effector or reporter groups. This conjugation method provides in high yields conjugates of high purity and homogeneity that are without inter-chain cross-linking and inactivated linker residues.

Description

Yoke closes method
The priority of the U.S. Provisional Application that the application requires to submit on June 3rd, 2009 number 61/183,774, its complete disclosure is clearly incorporated at this by reference.
Invention field
The present invention relates to make for example antibody or its fragment new method of closing of effect group (for example, cytotoxic agent) or reporter group (for example, radioactive label) and cell node mixture through difunctionality connector yoke.More specifically; (for example the present invention relates to make the effect group; Maytansinoid) or reporter group (for example; Radioactive label) with cell node mixture (for example antibody or its fragment) through the new method that difunctionality connector yoke closes, make this process eliminate to cause because the step of hydrolysate class of not expecting that intramolecularly or intermolecular reaction generate or the cross-linking agent class generation do not expected.
Background of invention
The cell node mixture for example antibody and effect group for example the conjugates of minicell toxic agents or cytotoxic protein causing great interest (Richart, A.D. and Tolcher, A.W. aspect the exploitation anticancer therapeutic agent; 2007; Nature Clinical Practice, 4,245-25).These conjugatess are because to the high specific of the selected antibody of antigen of expressing on the cell surface of tumor cell but tumour-specific.When specificity is bonded to tumor cell, antibody-cytotoxic agent conjugates by in dissolve target cancerous cell and degraded therein, discharge thus and suppress the for example competent cell toxic agents of microtubule kinetics or dna replication dna of necessary cell function, cause killing and wounding of cancerous cell.Adopted various connectors to connect antibody and cytotoxic agent, purpose be when strengthening internalization intracellular matter send the processing with conjugates, keep the stability of conjugates simultaneously in the blood plasma desired.These connectors comprise be designed the disulphide connector that has in various degree sterically hindered and influence its intramolecularly mercaptan reduction kinetics, cleavable the peptide connector for example valine-citrulline connect with cleavable connector not for example thioether be connected (Widdison; W. etc.; J.Med.Chem.; 2006,49,4392-4408; Erickson, H. etc., Cancer Res., 2006,66,4426-4433).
Cancer therapy that the cell node mixture for example use by the conjugates of antibody and labelling or the reporter gene tumor imaging that is used for the cancer patient, radionuclide-part conjugates is used, used in the immunoassay of diagnosis of various disorders and purifying biological the activating agent for example affinity chromatography of albumen, peptide and oligonucleotide are used.The labelling or the reporter gene that close with cell node mixture yoke comprise fluorogen and affinity labeling biological example element.
For example antibody (Ab) and effect group be (for example for the cell node mixture that connects via unreducible connection (for example thioether connection); Cytotoxic agent) or reporter group (for example; Radioactive label) conventional yoke closes method and adopts two different antibody response steps, and must use purification step.In first reactions step, antibody and the Heterobifunctional connector reaction that has two differential responses property groups (for example, X and Y).For example; In one approach, the reactive residue of antibody (for example lysine amino residue) causes the one or more reactive residues (for example lysine amino residue) at antibody to locate to add the connector with Y reactive group with the reaction of the X reactive group (for example N-hydroxy-succinamide ester) of different two difunctionality reagent.The initial antibody product of modifying must be from excessive connector or hydrolysis connector reagent purification before next step can take place.In second reactions step, contain antibody that the connector of Y reactive group (for example maleimide or Haloacetamide) modifies with contain reactive group for example mercaptan effector for example effect group (C) (for example, cytotoxic agent) reaction generate antibody-effector conjugates; Its in the additional purification step once more purification (referring to for example, United States Patent (USP) 5,208; 020,5,416,064 or 5; 024,834).Therefore, in above method, need at least two purification steps.
Comprise another kind of method that two reactions and purification step close antibody and effect group or reporter group with yoke utilize mercaptan residue in the antibody (through generate with mercaptan reagent for example 2-imido grpup sulfane modified antibodies or add the non-natural cysteine residues or produce through mutation through reducing natural disulfide bond) with the same difunctionality connector Y-L-Y reaction that contains Y reactive group (for example maleimide or Haloacetamide).
In antibody or peptide, add reactive group Y for example the major defect of maleimide (or Haloacetamide) be that natural histidine in reactive maleimide (or Haloacetamide) group and antibody or the peptide, lysine, tyrosine or cysteine residues experience intramolecularly or intermolecular reaction (Papini; A. etc.; Int.J.Pept.Protein Res.; 1992,39,348-355; Ueda, T. etc., Biochemistry, 1985,24,6316-6322), and the aqueous inactivation of Y maleimide base group.Before reacting with second of effect group or reporter group C, the aqueous inactivation of the intramolecularly of not expecting of natural histidine, lysine or cysteine residues or intermolecular reaction and Y maleimide base group produces crosslinking protein or heterogeneity conjugates and reduces the efficient of reacting with second of effect group or reporter group C in the maleimide that adds in the antibody (or Haloacetamide) group Y and the antibody.The homogeneous yoke of the crosslinking protein of heterogeneity conjugates product-produce owing to the not expected response of natural group (for example histidine, lysine, tyrosine or cysteine) in the initial group Y (for example maleimide base group) that adds and antibody or the peptide or non-activity maleimide residue that the aqueous inactivation produces or peptide-possibly have than expectation closes the active and stable of product difference.
Described through the disulfide bond yoke before and closed antibody and the method that contains the mercaptan cytotoxic agent (referring to for example, United States Patent (USP) 5,208,020,5,416,064,6,441,163, U.S. Patent Publication 2007/0048314 A1).These methods comprise the initial reaction of antibody and Heterobifunctional reagent, subsequently with second reaction that contains the mercaptan cytotoxic agent.At United States Patent (USP) 6; 441; Among 163 B1 optional method has been described; Wherein the reactive ester of the disulphide of cytotoxic agent connection is at first by purification, and then with antibody response, but this method comprised from containing extra reaction and the purification step that the mercapto cytotoxic agent begins before the antibody response step.
Other shortcomings that prepare at present the method for cell node mixture conjugates are two purification steps of needs, and these two purification steps have reduced gross production rate, and make this method for amplifying production, become loaded down with trivial details and uneconomical.
In view of the above; This area needs the modification method of development and preparation cell node mixture-drug conjugates compositions, and said cell node mixture-drug conjugates compositions has time and the cost that high-purity very and its preparation do not need loaded down with trivial details step and reduced the user.The invention provides this method.Of the present invention these will become obvious according to the invention description that this paper provides with other advantages and other inventive features.
Summary of the invention
The yoke that the invention describes the conjugates that the unreducible thioether that is used to prepare formula C-L-CBA connects closes method; Wherein C represents effector molecule or reporter molecule (for example, cytotoxic agent or radioactive label), and L is a connector; CBA be the cell node mixture (for example; Antibody or its fragment), (for example contain mercaptan cytotoxic agent (for example, maytansinoid) and XOR through utilization with difunctionality reagent; Cleavable or can not cracked connector) direct reaction; Mix unpurified reactant mixture and cell node mixture (for example, antibody or its fragment) subsequently, thus through more effectively, high yield and the technology of amplifying easily produces the conjugates that unreducible thioether connects.Another important advantage is the unreducible conjugates that this yoke closes the thioether connection of method no interchain protein-crosslinking of generation or inactivation residue (for example, maleimide or Haloacetamide residue).The disclosed new method of this description can be used for preparing any conjugates of being represented by following formula.
The accompanying drawing summary
Fig. 1 has shown antibody and maytansinoid DM1 (or DM4) and maleimide-PEG nThe yoke of the reactant mixture of-NHS connector closes.
Fig. 2 shows the Ab-(PEG of the method preparation of using the present invention's description 4-Mal)-the reduction SDS-PAGE of DM4 conjugates and the conjugates that uses traditional two-stage process preparation.Each sample lane contains 10 μ g albumen; Gel is used Coomassie blue stain.Swimming lane 1 and 2 contains molecular weight marker.Swimming lane 3 contains the conjugates with 6.1DM4/Ab through traditional two-stage process preparation.Swimming lane 4 contains method preparation of describing through the present invention and the conjugates that contains 6.2DM4/Ab.
Fig. 3 has shown the Ab-(PEG of the method preparation of using the present invention's description 4-Mal)-the albumen LabChip electrophoresis of DM4 conjugates and the conjugates that uses traditional two-stage process preparation.A.Ab-(PEG 4-Mal)-the albumen LabChip electrophoresis (Agilent 2100 Bioanalyzer/Agilent Proteins 230 test kits) of DM4 conjugates under reducing condition.Swimming lane 1: molecular weight marker thing; Swimming lane 2: the synthetic Ab-PEG of method that uses the present invention to describe 4-Mal-DM4,6.2D/Ab; Swimming lane 3: use two step yokes to close the synthetic Ab-PEG of method 4-Mal-DM4,6.1D/Ab; Swimming lane 4: the Ab (every swimming lane 0.24 microgram total protein) that closes of yoke not.The external markers that higher label, system peak and low label band representative are added from test kit.B. from the electrophoretic protein band of albumen LabChip quantitatively.
Fig. 4 has shown the Ab-(PEG of the method preparation of using the present invention's description 4-Mal)-MS of DM4 conjugates and the conjugates that uses traditional two-stage process preparation.A. the MS of the conjugates through the preparation of traditional two-stage process with 6.1DM4/Ab.Because the obvious inhomogeneity of conjugates, the MS peak can not good discrimination.B. the method for describing through the present invention prepares and contains the MS of the conjugates of 6.1DM4/Ab.Because the homogeneity of conjugates, the MS peak is by good discrimination.
Fig. 5 has shown the anti-CanAg antibody-PEG with 6.7DM1/ antibody 4Combination and unmodified antibody the combining expression CanAg antigenic COLO205 cell of-Mal-DM1 conjugates (the method preparation of using the present invention to describe) to expressing the antigenic COLO205 cell of CanAg.Being used in combination flat fluorescent measures.
Fig. 6 has shown the anti-CanAg antibody-PEG with 6.7DM1/ antibody 4-Mal-DM1 conjugates (the method preparation of using the present invention to describe) is to expressing the antigenic COLO205 cells in vitro of CanAg cytotoxicity.Conjugates is added into the COLO205 cell, after hatching 5 days continuously with conjugates, uses the WST-8 algoscopy to measure cell survival.Use anti-CanAg antibody blocking conjugates that excessive not yoke closes that the combination and the cytotoxicity of target cancerous cell are carried out the specificity of controlled trial with the explanation conjugates.
Fig. 7 shows that the yoke of the reactant mixture of antibody and DM1 (or DM4) and maleimide-sulfo group-NHS connector closes.
Fig. 8 shows the Ab-that uses the method preparation that the present invention describes (the reduction SDS-PAGE of sulfo group-Mal)-DM1 conjugates and the conjugates that uses traditional two-stage process preparation.Each sample lane contains 10 μ g albumen; Gel is used Coomassie blue stain.Swimming lane 1 contains molecular weight marker.Swimming lane 3 and 5 contains method preparation of describing through the present invention and the conjugates that contains 3.6/Ab and 6.2DM4/Ab respectively. Swimming lane 2 and 4 contains the conjugates for preparing and contain respectively 4.0/Ab and 5.7DM1/Ab through traditional two-stage process.
Fig. 9 has shown the Ab-that uses the method preparation that the present invention describes (the albumen LabChip electrophoresis of sulfo group-Mal)-DM1 conjugates and the conjugates that uses traditional two-stage process preparation.A.Ab-(sulfo group-Mal)-the albumen LabChip electrophoresis (Agilent 2100 Bioanalyzer/Agilent Proteins 230 test kits) of DM1 conjugates under reducing condition.Swimming lane 1: molecular weight marker thing; Swimming lane 2: the Ab that closes of yoke not; Swimming lane 3: use two step yokes to close the synthetic Ab-sulfo group-Mal-DM1 of method, 5.7D/Ab; Swimming lane 4: the synthetic Ab-sulfo group-Mal-DM1 of method that uses the present invention to describe, 5.6D/Ab; Every hole 0.22 microgram total protein.The external markers (every hole 0.24 microgram total protein) that higher label, system peak and low label band representative are added from test kit.B. from the electrophoretic protein band of albumen LabChip quantitatively.
Figure 10 has shown that the antibody of the method preparation of describing through the present invention-(LC-MS of sulfo group-Mal)-DM1 conjugates and the conjugates that closes the methods preparation through two step of tradition yokes relatively.The MS of the conjugates with 3.6DM1/Ab that the method for A. using the present invention to describe prepares shows the homogeneous conjugates with 1-6DM1 disengaging yoke compound peak.B. the MS that closes the conjugates with 4.0DM1/Ab of method preparation through traditional two step yokes shows corresponding to conjugates and has hydrolysis or the conjugates of the crosslinked connector (conjugates that for example has 2DM1; Add a L, two L and three 3L) the peak, show inhomogenous product.
Figure 11 has shown combination and unmodified antibody the combining expression CanAg antigenic COLO205 cell of anti-CanAg antibody-sulfo group-Mal-DM1 conjugates (the method preparation of using the present invention to describe) to expressing the antigenic COLO205 cell of CanAg with 5.6DM1/ antibody.In conjunction with measuring with flat fluorescent.
Figure 12 has shown that the anti-CanAg antibody-sulfo group-Mal-DM1 conjugates (the method preparation of using the present invention to describe) with 5.6DM4/ antibody is to expressing the antigenic COLO205 cells in vitro of CanAg cytotoxicity.Conjugates is added into the COLO205 cell, after hatching 5 days continuously with conjugates, uses the WST-8 algoscopy to measure cell survival.Use anti-CanAg antibody blocking conjugates that excessive not yoke closes that the combination and the cytotoxicity of target cancerous cell are carried out the specificity of controlled trial with the explanation conjugates.
Figure 13 shows that the yoke of the reactant mixture of antibody and DM1 (or DM4) and sulfo group-NHS SMCC connector closes.
Figure 14 shows the reduction SDS-PAGE of the method Ab-(SMCC)-DM1 conjugates for preparing and the conjugates that uses traditional two-stage process preparation that use the present invention to describe.Each sample lane contains 10 μ g total proteins; Gel is used Coomassie blue stain.Swimming lane 1 contains molecular weight marker, and swimming lane 2 contains the Ab that yoke not closes, and swimming lane 3 contains the conjugates with 3.1DM1/Ab through traditional two-stage process preparation, and swimming lane 4 contains the conjugates with 3.1DM1/Ab of the method preparation of describing through the present invention.
Figure 15 has shown the albumen LabChip electrophoresis of the method Ab-(SMCC)-DM1 conjugates for preparing and the conjugates that uses traditional two-stage process preparation that use the present invention to describe.The albumen LabChip electrophoresis (Agilent 2100 Bioanalyzer/Agilent Proteins 230 test kits) of A.Ab-SMCC-DM1 conjugates under reducing condition.Swimming lane 1: molecular weight marker thing; Swimming lane 2: the synthetic Ab-SMCC-DM1 of method that uses this patent to describe, 3.1D/Ab; Swimming lane 3: the Ab that closes of yoke not; Swimming lane 4: use two step yokes to close the synthetic Ab-SMCC-DM1 of method, 3.1D/Ab; (each swimming lane contains 0.24 microgram total protein).The external markers that higher label, system peak and low label band representative are added from test kit.B. from the electrophoretic protein band of albumen LabChip quantitatively.
The LC-MS that Figure 16 has shown the antibody of the method preparation of describing through the present invention-(SMCC)-DM1 conjugates and the conjugates that closes the methods preparation through two step of tradition yokes relatively.A. the MS of the conjugates through the preparation of continuous two-stage process with 3.1DM1/Ab.Because hydrolysis and the crosslinked segmental existence of connector, each main conjugates peak has the side peak of following.The MS of the conjugates that the method for B. describing through the present invention prepares with 3.1DM1/Ab.Because the homogeneity of conjugates, the MS peak is by good discrimination.
Figure 17 has shown the mechanism of interchain linkage and maleimide inactivation during the passing through traditional two-stage process yoke and closing of being proposed.
Figure 18 shows the Ab-that uses the method preparation that the present invention describes (the non-reduced SDS-PAGE of sulfo group-Mal)-DM4 conjugates and before the antibody yoke closes reaction, use the 4-maleimide butanoic acid quencher DM4 mercaptan (after initial DM4+NHS-sulfo group-Mal Heterobifunctional reagent coupling reaction) that dissociate.Each sample contains 10 μ g albumen; Gel is used Coomassie blue stain.Swimming lane 1 and 5 contains molecular weight marker.Swimming lane 2 contains independent Ab.Swimming lane 3 contains the conjugates of the method preparation of describing through the present invention, does not add 4-maleimide butanoic acid.Swimming lane 4 contains the conjugates of the method preparation of describing through the present invention, and (before the antibody yoke closes step) added 4-maleimide butanoic acid after initial DM4+NHS-sulfo group-Mal Heterobifunctional reagent.
Figure 19 has shown that the reactant mixture of use DM1 (or DM4) and SPDB connector prepares the antibody conjugates that disulphide is connected.
Figure 20 shows that the not purification reaction mixture yoke through antibody and DM1 (or DM4) and SPDB and NHS-PEG4-Mal connector closes to prepare and has disulphide and antibody-maytansinoid conjugates that can not cracked PEG4-Mal connector.
Figure 21 shows and to have disulphide and antibody-maytansinoid conjugates that can not cracked PEG4-Mal connector the MS of (the not purification reaction mixture yoke through antibody and DM1 or DM4 and SPDB and NHS-PEG4-Mal connector closes and prepares).
Figure 22 shows that the yoke of the reactant mixture of antibody and DM1 (or DM4) and SMCC connector closes.
Figure 23 shows the MS of the antibody-SMCC-DM1 conjugates of the method use SMCC preparation of describing through the present invention, and said conjugates contains average 3.1DM1/ antibody.
Figure 24 shows that the reactant mixture of use DM1 (or DM4) and SSNPB connector prepares the antibody conjugates that disulphide is connected.
The yoke of reactant mixture that Figure 25 shows antibody and DM1 (or DM4) and has a Heterobifunctional connector of aliphatic linear carbon chain closes.
Detailed Description Of The Invention
In detail with reference to certain embodiments of the present invention, the example is described with appended structure and formula now.Though the embodiment in conjunction with cited has been described the present invention, be appreciated that they are not will the present invention be limited to those embodiments.On the contrary, the present invention expection contain that all that possibly comprise in the scope of the invention that claims limit substitute, modification and equivalent.One skilled in the art will know that and can be used for embodiment of the present invention and those similar or be equal to many methods and materials described herein.
The invention describes make contain mercaptan the effect group (for example; Cytotoxic agent) or reporter group (for example; Radioactive label) with the cell node mixture (for example; Antibody) the yoke new method of closing, effector or the reporter molecule that wherein contains mercaptan are at first reacted in organic, aqueous or organic/aqueous mixed solvent with difunctionality connector reagent, and unpurified subsequently reactant mixture and cell node mixture react in organic, aqueous or organic/aqueous mixed solvent.
The legend abbreviation
The abbreviation of using in the description of following scheme and embodiment is:
C=effect group or reporter group (for example, cytotoxic agent or radioactive label)
L=connector (for example, cleavable or can not cracked connector)
X=amine reactive group (for example, N-hydroxy-succinamide ester (NHS ester), sulfo group-NHS ester, p-nitrophenyl phenolic ester, tetrafluoro sulphenyl ester, 1-hydroxyl-2-nitro-benzene-4-sulphonic acid ester)
Y=maleimide or Haloacetamide (iodoacetamide, acetbromamide)
Yb is the reactive disulfide group (for example, 2-pyridine radicals disulfide group, 4-pyridine radicals disulfide group, 2-nitro-pyridine radicals disulfide group, 5-nitro-pyridine radicals disulfide group, 2-carboxyl-5-nitro-pyridine radicals disulfide group) that mixes
X '=amido link
Y '=thioether (R-S-R ') or selenide (R-Se-R ') key
Yb '=two sulfur (R-S-S-R ') key
In one embodiment of the invention; The method that is used to prepare the conjugates that the cell node mixture is connected with the thioether of effector molecule or reporter molecule has been described; This method may further comprise the steps: the Heterobifunctional connector that a) makes formula X-L-Y and the effector molecule that contains thioether or reporter molecule C are (for example; Maytansinoid or radionuclide) in aqueous solvent, organic solvent or organic/aqueous hybrid reaction mixture, contact the intermediate product of production X-L-Y '-C; B) make reactant mixture need not purification and cell node mixture for example antibody (Ab) mix production Ab-(X '-L-Y '-C) mConjugates, wherein L is the unit (preferred 1-500 PEG sept, more preferably 1-24 PEG sept or more preferably 2-8 PEG sept) that has replacement or unsubstituted straight chain, side chain or cyclic alkyl, alkenyl or alkynyl, the simple or substituted aryl unit (substituent group is selected from alkyl, alkoxyl, halogen, nitro, fluoro, carboxyl, sulphonic acid ester, phosphate ester, amino, carbonyl, piperidyl) of 1-10 carbon atom or contain Polyethylene Glycol; X and Y are amine or thiol-reactive group for example N-hydroxy-succinamide ester and maleimide or Haloacetamide; Ab is an antibody; M is the integer of 1-20; X ' is the X site of modifying during with antibody response (for example, amido link); Y ' is the Y site of modifying during with the cytotoxic agent of for example effect group or reporter group or radioactive label reaction (for example, thioether bond); And c) through tangential flow filtration, dialysis or chromatograph (for example, gel filtration, ion exchange chromatography, hydrophobic interaction chromatograph) or its combination purification conjugates.Preferably, Y is the thiol-reactive group that is selected from maleimide or Haloacetamide.Preferably, L is the straight or branched alkyl with 1-6 carbon or 2-8 PEG sept.Preferably, C is the cytotoxic agent that is selected from maytansinoid, CC-1065 analog, taxane, DNA bonding agent, and more preferably, it is a maytansinoid.
The reaction process of expression in the formula 1 and 2:
X-L-Y+C→X-L-Y′-C (1)
Ab+X-L-Y '-C (from reacting 1 purification not) → Ab-(X '-m (2) of L-Y '-C)
Any purification that does not relate to intermediate product X-L-Y '-C; Therefore and provide and it has been mixed directly with antibody (unpurified intermediate product is added into antibody; Perhaps antibody is added into unpurified intermediate product) advantage; Close thereby make this method help yoke, because this does not need loaded down with trivial details purification step.Importantly, different with inactivation maleimide residue with the interchain protein-crosslinking of finding in the conjugates for preparing through traditional two-step reaction and purification procedures, this method produces the homogeneous conjugates that does not have interchain protein-crosslinking or inactivation maleimide residue.
Reaction 1 can high concentration Heterobifunctional connector, X-L-Y and effect group or reporter group C in aqueous solvent, organic solvent or organic/aqueous reaction mixture, carry out, cause than reaction rate faster during low concentration in the aqueous solution of the conjugates for preparing through traditional two-step reaction and purification procedures.
The intermediate product X-L-Y '-C that produces in the reaction 1 can suitably (for example hang down pH by unpurified freezing state; The aqueous solvent of pH～4-6), organic solvent or blended organic/aqueous mixture in or preserve the period that prolongs with lyophilised state; And can mix afterwards with antibody-solutions under, carry out final yoke and close reaction, increase the convenience of this reaction process thus with higher pH value at about 4-9.Intermediate product can be before mix with the cell node mixture on demand with the mixture diluted of organic solvent or aqueous buffer solution or organic solvent and aqueous buffer solution.The term " pact " that combines numerical value to use at this paper should be understood that to refer to all these type of numerical value, comprises from its all numerical value and little variation.The reaction of intermediate product X-L-Y '-C and antibody can be carried out under following pH value: about 4 to about pH9, preferred about 5 to 8.7 pH scope, more preferably from about 6.5 to about 8.5 pH scope, for example pH 6.5,6.6,6.7,6.8,6.9,7.0,7.1,7.2,7.3,7.4,7.5,7.6,7.7,7.8,7.9,8.0,8.1,8.2,8.3,8.4 and 8.5, pH scope wherein or its little variation.Being used for the buffer that antibody and intermediate product X-L-Y '-C react under about 6.5 to about 8.5 preferred pH scope is pK aNear the buffer of value this pH scope, for example phosphate buffer and HEPES buffer.These preferred buffer should not have primary amino radical or secondary amino group or can with other reactive groups of connector X (for example N-hydroxy-succinamide ester) reaction.
In first reaction, use than Heterobifunctional connector X-L-Y stoichiometry or excessive slightly C and reacted with all Y groups (for example maleimide) before guaranteeing adding not purified mixture to antibody.The optional extra process that can use quencher reagent (for example 4-maleimide butanoic acid, 3-maleimide propanoic acid or N-ethyl maleimide or iodoacetamide or iodoacetamide propanoic acid) with guarantee any unreacted C before mixing with antibody by quencher to minimize any undesirable exchange reaction of mercaptan-disulphide and natural antibody disulfide group.With the charged mercaptan quencher of polarity reagent (for example 4-maleimide butanoic acid or 3-maleimide propanoic acid) quencher the time, excessive unreacted C is converted to the charged adduct of polarity, and it can easily separate with covalently bound conjugates.Randomly, final reacting mixture before the purification with nucleophile for example contain amino nucleophile (for example, lysine, taurine, azanol) reaction with any unreacted connector of quencher (X-L-Y '-C).
The optional method that is used for the unpurified initial reaction mixture reaction of antibody and maytansinoid (DMx) and Heterobifunctional connector comprises the initial reaction mixture of DMx and Heterobifunctional connector is being mixed (when the DMx-connector reacts end) down with antibody at low pH (pH～5), add subsequently buffer agent or alkali with increase pH extremely about 6.5-8.5 close reaction to carry out yoke.
This new method is used to prepare the conjugates of antibody and cytotoxicity maytansinoid medicine.Based on the evaluation of reduction SDS-PAGE, albumen LabChip electrophoresis and mass spectrum, use the antibody-maytansinoid conjugates of this method preparation of in reaction process 1-2, listing aspect homogeneity, to be much better than conjugates unexpectedly through traditional two-step reaction and purification procedures preparation to conjugates.The yoke that comprises the present invention's description of reaction process 1-2 closes method without any need for the intermediate purification step, and therefore significantly more convenient than traditional two-stage process.
In second embodiment of the present invention; Described and be used to prepare the cell node mixture is connected conjugates with the thioether of effector molecule or reporter molecule method; This method may further comprise the steps: the same difunctionality connector of formula Y-L-Y is contacted in aqueous solvent, organic solvent or organic/aqueous hybrid reaction mixture to produce Y-L-Y '-C with effect group that contains mercaptan or amine or reporter group C (for example cytotoxic agent); B) reactant mixture need not purification and for example antibody in aqueous solution or aqueous/organic mixture, mix production Ab-(X '-conjugates of m of L-Y '-C), wherein L such as preceding text define; Y is mercaptan or amine reactive group for example maleimide or Haloacetamide, or N-hydroxyl sulfenimide or sulfo group N-hydroxyl sulfenimide; Ab is an antibody; M is 1 to 20 integer; Y ' be the Y site modified during with antibody response (for example; Thioether bond or amido link) or the Y site modified during with cytotoxic agent or effect group or reporter group reaction is (for example; Thioether bond or amido link); And c) through tangential flow filtration, dialysis or chromatograph (for example, gel filtration, ion exchange chromatography, hydrophobic interaction chromatograph) or its combination purification conjugates.The reaction process of expression in the formula 3 and 4:
Y-L-Y+C→Y-L-Y′-C (3)
Ab+Y-L-Y '-C (from reacting 3 purification not) → Ab-(Y '-L-Y '-C) m(4)
Any purification that does not relate to intermediate product Y-L-Y '-C, and be that favourable yoke closes method therefore.
In the 3rd embodiment, the method that is used to prepare the conjugates that the cell node mixture is connected with the disulphide of effector molecule or reporter molecule has been described, this method may further comprise the steps: a) make formula X-L-Y bHeterobifunctional connector and effect group or reporter group C (for example cytotoxic agent) in aqueous solvent, organic solvent or organic/aqueous hybrid reaction mixture, contact to produce intermediate product X-L-Y b'-C; B) make reactant mixture need not purification and antibody in aqueous solution or aqueous/organic mixture, mix production Ab-(X '-L-Y b'-C) mConjugates, wherein L such as preceding text define; Y bBe for example pyridyl disulfide or nitropyridine based bisulfide of reactive disulphide; X is for example N-hydroxy-succinamide ester or a sulfo group N-hydroxy-succinamide ester of amine reactive group; Ab is an antibody; M is 1 to 20 integer; X ' is the X site of modifying during with antibody response (for example amido link); Y bBe the Y that modifies during with cytotoxic agent or effect group or reporter group reaction bSite (disulphide); And c) through tangential flow filtration, dialysis or chromatograph (for example, gel filtration, ion exchange chromatography, hydrophobic interaction chromatograph) or its combination purification conjugates.Reaction process is represented with formula 5 and 6:
X-L-Y b+C→X-L-Y b′-C(5)
Ab+X-L-Y b'-C (from reacting 5 purification not) → Ab-(X-L-Y b'-C) m(6)
In the 4th embodiment; Described (disulfide bond) of connector through two types-can not cracked (thioether bond) and cleavable-prepared the method for antibody and effect group or reporter group conjugates; This method may further comprise the steps: a) make X-L-Y contact the intermediate compound with production X-L-Y '-C and X-L-Yb '-C with cytotoxic agent C with the X-L-Yb connector, b) shown in reaction equation 7-9, make reactant mixture need not purification with the antibody order or mix simultaneously:
X-L-Y+C→X-L-Y′-C (7)
X-L-Yb+C→X-L-Yb′-C (8)
Ab+X-L-Y '-C+X-L-Yb '-C (from reaction 7-8 purification not) → Ab-(X '-m of L-Y '-C) (X '-m ' (9) of L-Yb '-C)
With provide conjugates Ab-(X '-m of L-Y '-C) (X '-m ' of L-Yb '-C), wherein the definition of X, L, Y ', C, Yb ' and m such as preceding text provide, and m ' is 1 to 20 integer; And c) through tangential flow filtration, dialysis or chromatograph (for example, gel filtration, ion exchange chromatography, hydrophobic interaction chromatograph) or its combination purification conjugates.These two connector effect intermediate (X-L-Y '-C and X-L-Yb '-C) need not purification to be mixed with antibody with different order (at first X-L-Y '-C, X-L-Yb '-C then, perhaps at first X-L-Yb '-C, X-L-Y '-C then, perhaps simultaneously) and different proportion.
Reaction 1,3,5 and 7-8 can high concentration difunctionality connector (X-L-Y, X-L-Yb or Y-L-Y) and effect group or reporter group C in aqueous solvent, organic solvent or organic/aqueous reaction mixture, carry out; Cause than reaction rate faster during low concentration in the aqueous solution of the conjugates through the preparation of traditional two-step reaction and purification procedures, wherein the agent dissolves degree is restrictive.
In reaction 1,3,5 and 7-8 intermediate product X-L-Y '-C of producing or Y-L-Y '-C or X-L-Yb '-C can unpurified freezing state the aqueous solvent of suitably low pH, organic solvent or blended organic/aqueous mixture in or preserve the period that prolongs with lyophilised state; And can mix with antibody-solutions to carry out final yoke afterwards and close reaction, increase the convenience of this reaction process thus.
In first reaction, use than Heterobifunctional connector X-L-Y or Y-L-Y or X-L-Yb stoichiometry or excessive slightly C and reacted with all Y groups (for example maleimide) before guaranteeing adding not purified mixture to antibody.The optional extra process that can use quencher reagent (for example 4-maleimide butanoic acid or 3-maleimide propanoic acid or N-ethyl maleimide or iodoacetamide or iodoacetic acid) with guarantee any unreacted radical (for example mercaptan) before being added into antibody by quencher to minimize any undesirable exchange reaction of mercaptan-disulphide and natural antibody disulfide group.After C and difunctionality connector primary response, with the excessive C of charged polarity mercaptan quencher reagent quencher, make excessive C change into high polar water-soluble addition thing, it separates with covalently bound conjugates through gel filtration, dialysis or TFF easily.Final yoke closes the C that product does not contain any non-covalent connection.Randomly; Final reacting mixture 2,4,6 and 9 before the purification with nucleophile for example contain amino nucleophile (for example, lysine, taurine, azanol) handle with any unreacted connector of quencher (X-L-Y '-C, Y-L-Y '-C or X-L-Yb '-C).
Antibody and the optional method of the reaction of the not purification initial reaction mixture of DMx and difunctionality connector comprise the initial reaction mixture of DMx and Heterobifunctional connector is being mixed (when the reaction of DMx-connector finishes) down at low pH (pH～5) with antibody, add subsequently buffer agent or alkali with increase pH extremely about 6.5-8.5 close reaction to carry out yoke.
Through will need not purification ground order derived from two or more connectors-effector intermediate of two or more different effect things or be added into antibody simultaneously, a plurality of copies that surpass a kind of type effects thing can close with the antibody yoke.
The effect group
The term " effect group " that the interchangeable use of term effect group or effector molecule and this paper use or " effector molecule " meaning are to comprise cytotoxic agent.In some aspects, possibly hope that effect group or effector molecule connect to reduce potential steric hindrance through the spacerarm of all lengths.The a plurality of copies that surpass a kind of effector can be through to the antibody order or add simultaneously that unpurified yoke is bonded to antibody derived from the two or more connectors-effector intermediate of two or more different effect.
Can be used for the analog that cytotoxic agent of the present invention comprises chemotherapeutics or chemotherapeutics." chemotherapeutics " is the chemical compound that is used to treat cancer.The instance of chemotherapeutics comprises alkylating agent, for example thiotepa and cyclophosphamide (CYTOXAN TM); Alkyl sulfonate esters is busulfan, an improsulfan and A-20968 for example; Aziridine is benzodopa, carboquone, meturedopa and uredopa for example; Aziridine and methyl melamine comprise altretamine, triethylenemelamine, triethylene phosphoramide (TEPA), triethylene thiophosphoramide and trimethyl tripolycyanamide; Acetogenin (particularly bullatacin and bullatacinone); Camptothecine (comprising synthetic similar hycamtin); Bryostatin; Callystatin; CC-1065 (comprising its adozelesin, carzelesin and bizelesin synthetic analogues); Cryptophycin (particularly cryptophycin 1 and cryptophycin 8); Dolastatin; Times carcinomycin (comprising synthetic analogues KW-2189 and CBI-TMI); Soft coral alcohol; Pancratistatin; A Corallium Japonicum Kishinouye alcohol of crawling; The sponge inhibin; Chlormethine, for example chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, chlormethine, Mechlorethaminoxide Hydrochloride, melphalan, novoembichin, phenesterine, PM, trofosfamide, uracil mustard; Nitroso ureas, for example Carmustine, chlorozotocin, Fotemustine, lomustine, nimustine, Ranimustine; Antibiotic, for example (for example, calicheamicin, particularly calicheamicin γ 1 and calicheamicin θ I are referring to for example Angew Chem Intl.Ed.Engl.33:183-186 (1994) for the enediyne antibiotic; Dynemicin comprises dynemicin A; The Ai Sibo mycin; And neocarzinostain NCS chromophore and relevant chromoprotein enediyne antibiotic chromophore chromomophores), aclacinomysins, D actinomycin D, authramycin, azaserine, bleomycin, actinomycin C, carabicin, carminomycin, cardinophyllin; Chromomycin; Actinomycin D; Daunoblastin; Detorubicin; 6-diazonium-5-oxygen-L-nor-leucine; Amycin (comprises morpholino-amycin; Cyanic acid morpholino-amycin; 2-pyrrolin generation-amycin and deoxidation amycin); Epirubicin; Esorubicin; DMDR; Marcellomycin; Nitomycins; Mycophenolic acid; Nogalamycin; Olivomycin; NK-631; Potfiromycin; Puromycin; Triferricdoxorubicin; Rodorubicin; Rufocromomycin; Streptozocin; Tubercidin; Ubenimex; Neocarzinostain NCS; Zorubicin; Antimetabolite, for example methotrexate and 5-fluorouracil (5-FU); Folacin, for example 9,10-dimethylpteroylglutamic acid, methotrexate, pteroyltriglutamic acid, Trimetrexate; Purine analogue, for example fludarabine, Ismipur, ITG, thioguanine; Pyrimnidine analog, for example NSC-145668, azacytidine, 6-azauridine, carmofur, cytosine arabinoside, two BrdU, doxifluridine, enocitabine, 5 fluorodeoxyuridines, 5-FU; Androgen, for example calusterone, dromostanolone propionate, epitiostanol, Mepitiostane, testolactone; Anti-adrenal gland, for example aminoglutethimide, mitotane, trilostane; Folic acid supplement, for example folinic acid; Aceglatone; The aldophosphamide glucosides; Amino-laevulic acid; Amsacrine; Bestrabucil; Bisantrene; Edatraxate; Defofamine; Demecolcine; Diaziquone; Elfomithine; Elliptinium Acetate; Epoxy gathers tubulin; AY-62013; AY-62013; Hydroxyurea; Lentinan; Lonidamine; Maytansinoid, for example maytansine and ansamitocin; Methyl GAG; Mitoxantrone; Mopidamol; C-283; Pentostatin; Phenamet; Perarubicin; Podophyllinic acid; 2-ethyl hydrazides; Procarbazine; PSK
Figure BPA00001479440100171
Tetrahydroform; Rhizomycin; Sizofiran; All spiral shell amine; Tenuazonic acid; Triaziquone; 2,2 ', 2 " trichloro-triethylamines; Trichothecene family toxin (particularly T-2 toxin, verracurin A, Roridine A and anguidine); Urethanes; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitolactol; Pipobroman; Gacytosine; Galactoside (" Ara-C "); Cyclophosphamide; Thiotepa; Taxane, for example paclitaxel (TAXOL
Figure BPA00001479440100172
Bristol-Myers Squibb Oncology, Princeton, NJ.) and Docetaxel (TAXOTERE
Figure BPA00001479440100173
Rhone-Poulenc Rorer, Antony, France); Chlorambucil; Gemcitabine; The 6-thioguanine; Purinethol; Methotrexate; Platinum analogs, for example cisplatin and carboplatin; Vincaleucoblastine; Platinum; Epipodophyllotoxin (VP-16); Ifosfamide; Ametycin; Mitoxantrone; Vincristine; Vinorelbine; Nvelbine; Novantrone; Teniposide; Daunomycin; Aminopterin; Xeloda; Ibandronate; CPT-11; Topoisomerase enzyme inhibitor RFS 2000; DFMO (DMFO); Tretinoin; Capecitabine; With the acceptable salt of above any pharmacy, acid or derivant.This definition also comprise act as regulate or inhibitory hormone to the antihormone agent of function of tumor; For example estrogen antagonist comprises for example tamoxifen, raloxifene, aromatase inhibition 4 (5)-imidazoles, 4-hydroxy tamoxifen, trioxifene, keoxifene, LY117018, onapristone and toremifene (Fareston); And androgen antagonist, for example Drogenil, nilutamide, bicalutamide, leuprolide and goserelin; SiRNA and the acceptable salt of above any pharmacy, acid or derivant.Can be used for other chemotherapeutics of the present invention and be disclosed in US publication 20080171040 or US publication 20080305044, and integral body is incorporated into by reference.
In a preferred embodiment, the agent of chemotherapy cytotoxic is the micromolecule cytotoxic agent basically." small-molecule drug " is widely used at this paper that finger for example can have 100 to 1500, is more suitable for 120 to 1200, preferred 200 to 1000 molecular weight and have organic compound, inorganic compound or the organo-metallic compound less than about 1000 molecular weight usually.Micromolecule cytotoxic agent of the present invention comprises oligopeptide and the other biological molecule that has less than about 1000 molecular weight.The micromolecule cytotoxic agent in this area by well-characterized, for example in WO05058367A2, European Patent Application No. 85901495 and 8590319, and at U.S. Patent number 4,956,303 and other in, its by reference integral body incorporate into.
Preferred micromolecule cytotoxic agent is to allow to be connected to those of cell node mixture.The present invention includes known cytotoxic agent and can become known those.Preferred especially micromolecule cytotoxic agent comprises cytotoxic agent.
Cytotoxic agent can be to cause cell death or inducing cell death or reduce any chemical compound of cell survival with certain mode, and wherein every kind of cytotoxic agent comprises thiol moiety.
Preferred cytotoxic agent is maytansinoid chemical compound, taxane compounds, CC-1065 chemical compound, daunoblastin chemical compound and amycin chemical compound, pyrrolo-Benzodiazepine dimer, calicheamicin, auristatin and analog and derivant, and wherein some are described hereinafter.
Be not necessarily micromolecular other cytotoxic agents for example siRNA be also included within the scope of the invention.For example, siRNA can be connected to cross-linking agent of the present invention (disclosing 20050107325 and 20070213292 referring to for example United States Patent (USP)) through the method that is usually used in modified oligonucleotide.Therefore, with its 3 ' or 5 '-siRNA of phosphoramidite form and have hydroxy functional group cross-linking agent end reaction and between siRNA and cross-linking agent, produce ester bond.Similarly, the siRNA phosphoramidite causes cross-linking agent to be connected through amine with siRNA with the cross-linking agent reaction that has terminal amino group.SiRNA is described in detail in U.S. Patent Publication: 20070275465,20070213292,20070185050,20070161595,20070054279,20060287260,20060035254,20060008822,20050288244,20050176667, its this by reference integral body incorporate into.
Maytansinoid
It is well known in the art can be used for maytansinoid of the present invention, and can separate or prepare according to known method is synthetic from natural origin according to known method.
The plain alkaloidal instance of the pass DENGMU that is fit to comprises maytansinol and maytansinol analog.The instance of the maytansinol analog that is fit to comprise aromatic ring with modification those with have those that other positions modify.
The instantiation of the maytansinol analog that is fit to aromatic ring of modification comprises:
(1) C-19-dechlorination (U.S. Patent number 4,256,746) (P2 prepares through LAH reduction maytenin);
(2) C-20-hydroxyl (or C-20-demethylation)+/-C-19-dechlorination (U.S. Patent number 4,361,650 and 4,307,016) (through using streptomycete or actinomycetes demethylation or using the LAH dechlorination to prepare); With
(3) C-20-demethoxylation, C-20-acyloxy (OCOR) ,+/-dechlorination (U.S. Patent number 4,294,757) (preparing) through using the acid chloride acidylate.
Instantiation with maytansinol analog that is fit to of modifying other positions comprises:
(1) C-9-SH (U.S. Patent number 4,424,219) (react prepare through maytansinol and H2S or P2S5);
(2) C-14-alkoxy methyl (demethoxylation/CH20R) (U.S. Patent number 4,331,598);
(3) C-14-hydroxymethyl or acyloxy methyl (CH2OH or CH2OAc) (U.S. Patent number 4,450,254) (from the Nocardia preparation);
(4) C-15-hydroxyl/acyloxy (U.S. Patent number 4,364,866) (transforming maytansinol through streptomycete prepares);
(5) C-15-methoxyl group (U.S. Patent number 4,313,946 and 4,315,929) (separating) from trewianudiflora;
(6) C-18-N-demethylation (U.S. Patent number 4,362,663 and 4,322,348) (the maytansinol demethylation being prepared) through streptomycete; With
(7) 4,5-deoxidations (U.S. Patent number 4,371,533) (preparing) through titanous chloride ./LAH reduction maytansinol.
Be used for the plain alkaloidal synthetic full disclosure of sulfur-bearing of the present invention alcohol Folium Mayteni hookeri in U.S. Patent number 5,208,020,5,416,064 with Patent Application No. 20040235840.
It all is useful being expected at the maytansinoid that C-3 position, C-14 position, C-15 position or C-20 position have thiol moiety.The C-3 position is preferred, and the C-3 position of maytansinol is preferred especially.The maytansinoid of N-methyl-alanine-contain C-3 thiol moiety and N-methyl-cysteine-contain the maytansinoid of C-3 thiol moiety and the analog of each also is preferred.
The instantiation that is used for N-methyl-alanine of the present invention-contain maytansinoid derivant of C-3 thiol moiety is represented by formula M1, M2, M3, M6 and M7.
Figure BPA00001479440100201
Wherein:
L is 1 to 10 integer; And May is a maytansinoid.
Figure BPA00001479440100202
Wherein:
R 1And R 2Be H, CH 3Or CH 2CH 3, and can be identical or different;
M is 0,1,2 or 3; And
May is a maytansinoid.
Figure BPA00001479440100211
Wherein:
N is 3 to 8 integer; And
May is a maytansinoid.
Wherein:
L is 1,2 or 3;
Y 0Be Cl or H; And
X 3Be H or CH 3
Wherein:
R 1, R 2, R 3, R 4Be H, CH 3Or CH 2CH 3, and can be identical or different;
M is 0,1,2 or 3; And
May is a maytansinoid.
The instantiation that is used for N-methyl-cysteine of the present invention-contain maytansinoid derivant of C-3 thiol moiety is represented by formula M4 and M5.
Figure BPA00001479440100222
Wherein:
O is 1,2 or 3;
P is 0 to 10 integer; And May is a maytansinoid.
Wherein:
O is 1,2 or 3;
Q is 0 to 10 integer;
Y 0Be Cl or H; And
X 3Be H or CH 3
Preferred maytansinoid is to be disclosed in U.S. Patent number 5,208,020; 5,416,064; 6,333.410; 6,441,163; 6,716,821; RE39,151 and 7,276, those of 497.
Taxane
According to cytotoxic agent of the present invention also can be taxane.
Can be used for taxane of the present invention has been modified to and has contained thiol moiety.Be used for taxanes more of the present invention and have the formula T1 shown in following:
Figure BPA00001479440100241
Preferred taxane is to be disclosed in U.S. Patent number 6,340,701; 6,372,738; 6.436,931; 6,596,757; 6,706,708; 7,008,942; In 7,217,819 and 7,276,499 those.
The CC-1065 analog
According to cytotoxic agent of the present invention also can be the CC-1065 analog.
According to the present invention, the CC-1065 analog contains A subunit and B or B-C subunit.Preferred CC-1065 analog is to be disclosed in U.S. Patent number 5,475,092; 5,595,499; 5,846,545; 6,534,660; 6,586,618; 6,756,397 and 7,049, those of 316.
Daunoblastin/amycin analog
According to cytotoxic agent of the present invention also can be daunoblastin analog or amycin analog.
Daunoblastin of the present invention and amycin analog can be modified to and contain thiol moiety.Amycin/daunoblastin analog with modification of the present invention of thiol moiety is described in WO 01/38318.Amycin/daunoblastin of modifying can be synthetic according to known method (referring to for example, U.S. Patent number 5,146,064).
Auristatin comprises auristatin E, auristatin EB (AEB), auristatin EFP (AEFP), monomethyl auristatin E (MMAE) and is described in U.S. Patent number 5,635,483, Int.J.Oncol.15:367-72 (1999); Molecular Cancer Therapeutics, the 3rd volume, the 8th phase, pp.921-932 (2004); Patent Application No. 11/134826, U.S. Patent Publication 20060074008,2006022925.
Cytotoxic agent according to the present invention comprises pyrrolo-Benzodiazepine dimer known in the art (U.S. Patent number 7,049,311; 7,067,511; 6,951,853; 7,189,710; 6,884,799; 6,660,856).
Analog and derivant
Cytotoxic agent those skilled in the art understand easily, and each of cytotoxic agent described herein can be modified as follows: the chemical compound that obtains still keeps the specificity and/or the activity of initial compounds.The technical staff also understands, and these chemical compounds many can substitute cytotoxic agent described herein and use.Therefore, cytotoxic agent of the present invention comprises the analog and the derivant of chemical compound described herein.
Reporter group
The term " reporter group " that the interchangeable use of term reporter group or reporter molecule and this paper use or " reporter molecule " refer to send the material to predetermined substance or cell for diagnosis or therapeutic purposes through the affine part of the specificity of reagent; Instance has radiosiotope, paramagnetic contrast agent and anticarcinogen.Various labellings or reporter group are used for cancer patient's tumor imaging and use; The immunoassay of diagnosis of various disorders are used; Use the cancer therapy of radionuclide-part conjugates and be used for the for example affinity chromatography application of albumen, peptide and oligonucleotide of purifying biological activating agent.The labelling or the reporter group that close with cell node mixture yoke comprise fluorogen and affinity labeling biological example element.This reporter group can be with reference to US publication 2007/0092940.The reporter group that comprises biological example element or fluorescein can also be connected to PEG conjugates part.Many suitable reporter groups are known in the art, and for example U.S. Patent number 4,152,411 with Hirschfeld U.S. Patent number 4; 166,105, U.S. Patent number 5,223, and 242, U.S. Patent number 5; 501,952, the U.S. discloses 20090136940, and integral body is incorporated into by reference.
Connector
Conjugates can be through the in vitro method preparation.In order to connect medicine and cell node mixture, used linking group.The linking group that is fit to is well known in the art and comprises cleavable not or the connector of cleavable.Can not cracked connector be can be to stablize any chemical part that covalent manner connects cytotoxic agent and cell node mixture.Can not tolerate sour inductive cracking, photoinduced cracking, the inductive cracking of peptidase, the inductive cracking of esterase and disulfide bond cracking basically by cracked connector.Instance that can not cracked connector comprises having and is used for the N-succinimide ester of medicine, reporter group or the reaction of cell node mixture, N-thiosuccimide ester moiety, based on dimaleoyl imino-or the connector of halo acetyl group part.The cross-linking reagent that comprises based on dimaleoyl imino-part comprises N-succinimido 4-(maleimide ylmethyl) cyclohexane carboxylate (SMCC); " long-chain " analog (LC-SMCC) N-succinimido-4-(N-maleimide ylmethyl)-cyclohexane extraction-1-carboxyl-(the 6-amide alkyl caproate) of SMCC; κ-dimaleoyl imino hendecanoic acid N-succinimide ester (KMUA); γ-maleimide butanoic acid N-succinimide ester (GMBS); ε-dimaleoyl imino capric acid N-hydroxy-succinamide ester (EMCS); M-dimaleoyl imino benzoyl-N-hydroxy-succinamide ester (MBS); N-(α-dimaleoyl imino acetoxyl group)-succinimide ester (AMAS); Succinimido-6-(β-dimaleoyl imino propionamido-) alkyl caproate (SMPH); N-succinimido 4-(p-dimaleoyl imino phenyl)-butyrate (SMPB) and N-(p-dimaleoyl imino phenyl) isocyanates (PMPI).The cross-linking reagent that comprises based on halo acetyl group part comprises N-succinimido-4-(iodo acetyl group)-Aminobenzoate (SIAB), N-succinimido iodo acetas (SIA), N-succinimido bromacetate (SBA) and N-succinimido 3-(acetyl bromide is amino) propionic ester (SBAP).
Forming not, other cross-linking reagents that lack sulphur atom of cleavable connector also can be used for the present invention.This connector can be derived from the part based on dicarboxylic acids.The part that is fit to based on dicarboxylic acids include but not limited to following shown in the alpha, omega-dicarboxylic acid of general formula:
HOOC-X l-Y n-Z m-COOH
Wherein X is straight or branched alkyl, the alkenyl or alkynyl with 2 to 20 carbon atoms; Y is cycloalkyl or the cycloalkenyl group with 3 to 10 carbon atoms; Z be have replacement or the unsubstituted aromatic group of 6 to 10 carbon atoms or wherein hetero atom be selected from replacement or the unsubstituted heterocyclic of N, O or S; And wherein l, m and n respectively do for oneself 0 or 1, and condition is that l, m and n are not 0 simultaneously.
Many U.S. Patent Publication 20050169933 that are described in detail in of the disclosed not cleavable of this paper connector.
The connector of cleavable is the connector of cleavable under temperate condition, and said temperate condition i.e. the active impregnable condition of cytotoxic agent wherein.Many known connectors belong to this classification and describe hereinafter.
The unstable connector of acid is the connector of cleavable under acid pH.For example, for example endosome and lysosome have acid pH (pH 4-5) and the condition that is fit to the unstable connector of cracking acid are provided in some intracellular region chamber.
The photo-labile connector be used for body surface and a lot of light can and body cavity in.And, the penetrable tissue of infrared light.
Some connectors can be by the peptide enzymatic lysis.Only some peptide is easily in cell or the cracking of extracellular; Referring to for example Trouet etc., 79 Proc.Natl.Acad.Sci.USA, 626-629 (1982); 43 Int.J.Cancer such as Umemoto; 677-684 (1989) and lysosomal-hydrolase cleavable valine-citrulline linkage (valine-citrulline of lysosomal hydrolase cleavable is connected) (United States Patent (USP) 6,214,345 B1).And peptide is made up of a-amino acid and peptide bond, and peptide bond chemically is being the amido link between an amino acid whose carboxylic acid and another the amino acid whose alpha-amido.Think other amido links for example the key between the ε amino of carboxylic acid and lysine be not peptide bond and be can not be cracked.
Some connectors can be by the esterase cracking.Equally, only some ester can be by the esterase cracking of in the cell or extracellular existence.Ester is that carboxylic acid and pure condensation form.Simple ester is with for example aliphatic alcohol and circlet shape and the generation of little aromatic alcohol of simple alcohol.For example, the inventor finds there is not the esterase at the C-3 place cracking ester of maytansine, because the alkoxide component maytansinol of ester is very big and complicated.
Preferred cleavable connector molecule for example comprise N-succinimido 3-(2-pyridine radicals disulfide group) propionic ester (SPDP) (referring to for example, Carlsson etc., Biochem.J; 173:723-737 (1978)), N-succinimido 4-(2-pyridine radicals disulfide group) butyrate (SPDB) is (referring to for example United States Patent (USP) 4; 563,304), N-succinimido 4-(2-pyridine radicals disulfide group) valerate (SPP) (referring to for example, CAS registration number 341498-08-6) and other reactant cross-linkers; For example be described in United States Patent (USP) 6; In 913,748 those, it is incorporated at this by reference.
Can be used for other connectors of the present invention and comprise charged connector or hydrophilic connector, be described in Patent Application No. 12/433,604 and 12/433,668 respectively, it is incorporated at this by reference.
The cell node mixture
The cell node mixture that the present invention uses is albumen (for example, immunoglobulin and NIg), and it combines with target antigen specificity on the cancerous cell.These cell node mixture comprise:
-antibody comprises:
-surface treatment antibody (resurfaced antibody) (U.S. Patent number 5,639,641);
-humanization or fully human antibodies (humanization or fully human antibodies be selected from but be not limited to huMy9-6, huB4, huC242, huN901, DS6, CD38, IGF-IR, CNTO 95, B-B4, Qu Sizhu monoclonal antibody, than cut down pearl monoclonal antibody, sibrotuzumab and Rituximab (referring to for example, U.S. Patent number 5,639,641,5; 665,357 and 7,342; 110, International Patent Application WO 02/16,401, US publication 20060045877, US publication 20060127407, US publication 20050118183, Pedersen etc., (1994) J.Mol.Biol.235; 959-973, Roguska etc., (1994) Proceedings of the National Academy of Sciences, the 91st volume; 969-973, Colomer etc., Cancer Invest., 19:49-56 (2001), Heider etc.; Eur.J.Cancer, 31A:2385-2391 (1995), Welt etc., J.Clin.Oncol.; 12:1193-1203 (1994) and Maloney etc., Blood, 90:2188-2195 (1997)); With
-antibody fragment, for example sFv, Fab, Fab ' and F (ab ') 2, it preferentially combines target cell (Parham, J.Immunol.131:2895-2902 (1983); Spring etc., J.Immunol.113:470-478 (1974); Nisonoff etc., Arch.Biochem.Biophys.89:230-244 (1960));
Other cell node mixture comprise other cell node hop protein and polypeptide, and example is following but be not limited thereto:
-ankyrin repetitive proteins (DARPins; Zahnd etc., J.Biol.Chem., 281,46,35167-35175, (2006); Binz, H.K., Amstutz, P.& Pluckthun, A. (2005) Nature Biotechnology, 23,1257-1268) or ankyrin appearance repetitive proteins or synthetic peptide, be described in for example US publication 20070238667; U.S. Patent number 7,101,675; WO/2007/147213; WO/2007/062466);
-interferon (for example α, β, γ);
-lymphokine is IL-2, IL-3, IL-4, IL-6 for example;
-hormone, for example for example androgen and estrogen of insulin, TRH (throtropin releasing hormone), MSH (melanotropin), steroid hormone; With
-somatomedin and colony stimulating factor, for example EGF, TGF-α, IGF-1, G-CSF, M-CSF and GM-CSF (Burgess, Immunology Today 5:155-158 (1984)).
When the cell node mixture was antibody, its combined polypeptide antigen and can be for example somatomedin of transmembrane molecule (for example receptor) or part.Exemplary antigen comprises for example feritin of molecule; Growth hormone comprises human growth hormone and BGH; SRF; Parathyroid hormone; Thyrotropin; Lipoprotein; α-1-antitrypsin; INSULIN A-chain; Insulin B-chain; Proinsulin; Follicle stimulating hormone; Calcitonin; Lutropin; Glucagon; Thrombin, for example the vmc factor, the IX factor, tissue factor (TF) and the von Willebrands factor; Anticoagulin, for example PROTEIN C; Atrionatriuretic factor; Curosurf; Plasminogen activator, for example urokinase or Urina Hominis or histiotype plasminogen activation factor (t-PA); Bombesin; Thrombin; Hemopoietic growth factor; Tumor necrosis factor-alpha and-β; Enkephalinase; RANTES (regulate and activate normal T cellular expression and EF); Human macrophage inflammatory protein (MIP-1-α); Serum albumin, for example human serum albumin; The Muellerian inhibiting substances; Relaxin A-chain; Relaxin B-chain; Relaxin is former; Mice GTH related peptides; Microprotein, for example beta-lactamase; The DNA enzyme; IgE; Cytotoxic t lymphocyte-associated antigen (CTLA), for example CTLA-4; Inhibin; Activin; VEGF (VEGF); Hormone or growth factor receptors; Protein A or D; Rheumatoid factor; SDNF, for example bone source property SDNF (BDNF), neurotrophin-3 ,-4 ,-5 or-6 (NT-3, NT4, NT-5 or NT-6), or nerve growth factor, for example NGF-β.; Platelet-derived somatomedin (PDGF); Fibroblast growth factor, for example aFGF and bFGF; Epidermal growth factor (EGF); Transforming growth factor (TGF), for example TGF-α and TGF-β comprise TGF-β 1, TGF-β 2, TGF-β 3, TGF-β 4 or TGF-β 5; Insulin like growth factor-1 and-II (IGF-I and IGF-II); Des (1-3)-IGF-I (brain IGF-I), insulin-like growth factor binding protein; CD albumen, for example CD3, CD4, CD8, CD19, CD20 and CD40; Erythropoietin; Bone-inducing factor; Immunotoxin; BMP (BMP); Interferon, for example interferon-' alpha ' ,-β and-γ; Colony stimulating factor (CSF), for example, M-CSF, GM-CSF and G-CSF; Interleukin (IL), for example, IL-1 to IL-10; Superoxide dismutase; TXi Baoshouti; Surface membrane protein; Decay accelerating factor; Virus antigen, for example, the part of HIV peplos; Transport protein; Homing receptor; Addressin; Regulate albumen; Integrin, the for example α-V subunit of CD11a, CD11b, CD11c, CD18, ICAM, VLA-4 and VCAM, heterodimer human beta 2 integrin receptor; Tumor associated antigen, for example HER2, HER3 or HER4 receptor; Any one fragment with above-listed polypeptide.
The preferred antigens of the antibody that the present invention is contained comprises CD albumen, for example CD3, CD4, CD8, CD19, CD20, CD34 and CD46; ErbB receptor family member, for example EGF receptor, HER2, HER3 or HER4 receptor; Cell adhesion molecule, for example LFA-1, Mac1, p150.95, VLA-4, ICAM-1, VCAM, α 4/ β, 7 integrins and α v/ β 3 integrins comprise its α or β subunit (for example, anti-CD11a, anti-CD 18 or anti-CD11b antibody); Somatomedin, for example VEGF; Tissue factor (TF); TGF-β.; IFN-(α-IFN); Interleukin, for example IL-8; IgE; Blood group antigen Apo2, death receptor; The flk2/flt3 receptor; Fat (OB) receptor; The mpl receptor; CTLA-4; PROTEIN C etc.Here most preferred target is IGF-IR, CanAg, EGF-R, EphA2, MUC1, MUC 16, VEGF, TF, CD 19, CD20, CD22, CD33, CD37, CD38, CD40, CD44, CD56, CD138, CA6, Her2/neu, CRIPTO (albumen that in most people's breast cancer cell, produces with the level that raises), α v/ β 3 integrins, α v/ β 5 integrins, TGF-β, CD11a, CD 18, Apo2 and C24.
Monoclonal antibody technique allows to produce the specific cell bonding agent of monoclonal antibody form.This area is well known that through with the for example complete target cell of interested antigen, for example virus capsid protein immunized mice, rat, hamster or any other mammal produce the technology of monoclonal antibody from the intact virus of the isolating antigen of target cell, intact virus, attenuation and virus protein especially.Can also use people's cell of sensitization.Another method that produces monoclonal antibody be to use sFv (strand variable region), the particularly phage library of people sFv (referring to for example, Griffiths etc., U.S. Patent number 5,885,793; McCafferty etc., WO 92/01047; Liming etc., WO 99/06587.).
The selection of the cell node mixture that is fit to is according to the selection problem of treating the specific cells crowd of targeting, but generally speaking, if suitable a kind of be obtainable, it is preferred then preferentially combining monoclonal antibody of target cell and fragment thereof.
For example, monoclonal antibody My9 is the Mus IgG to the CD33 antigenic specificity that exists on acute myeloid leukemia (AML) cell 2aAntibody (Blood 77:2404-2412 (1991) such as Roy) and can be used for treating AML patient.Similarly, the anti-B4 of monoclonal antibody is Mus IgG 1, its combine on the B cell CD 19 antigens (Nadler etc., J.Immunol.131:244-250 (1983)) and when target cell be that this antigenic B cell of expression or diseased cells for example can be used when non-Hodgkin lymphoma or chronic lymphoblast leukemia.Similarly, antibody N901 is the Mus monoclonal IgG of the CD56 that exists on other tumor cells that combine small cell lung cancer cell and neuroendocrine to originate 1Antibody J Nat.Cancer Inst.88:1136-1145 (1996) such as () Roy, combine the antigenic huC242 antibody of CanAg, combine the Qu Sizhu monoclonal antibody of HER2/neu and combine the anti-EGF receptor antibody of EGF receptor.
Purification process
Conjugates of the present invention, i.e. end product, by purification to remove any unreacted or the not effector molecule that closes of yoke or reporter molecule or unreacted connector or the not connector of the yoke hydrolysis of closing.Purification process can be tangential flow filtration (also being called TFF, ultrafiltration or the diafiltration of cross-flow filtration), gel filtration, adsorption chromatography, selective precipitation or its combination.The adsorption chromatography method comprises ion exchange chromatography, hydroxylapatite chromatography, hydrophobic reactant chromatograph (HIC), dewatering electric charge inducing color chromatogram (HCIC), mixed type ion exchange chromatography, immobilization metal affinity chromatography (IMAC), dye ligand body colour spectrum, affinity chromatography and reversed phase chromatography.The conjugates Ab-that for example, describes in the formula 2 (X '-L-Y '-C) mConnector X-L-Y or X-L-Y '-C purification from unreacted C or unreacted/hydrolysis.Similarly, the conjugates of describing in the formula 4,6 and 9 is by purification.This purification process is well known by persons skilled in the art, and can be referring to for example US publication 2007/0048314.
The connector of the hydrolysis of not expecting in the conjugates or protein-crosslinking
Utilize albumen and traditional yoke method of closing of the primary response of the Heterobifunctional connector with reactive maleimide or Haloacetamide residue to have two major defects: (i) owing to the aqueous inactivation of the connector that adds in the antibody before reacting with effector molecule or reporter molecule, yoke closes product and possibly be made up of the connector of hydrolysis; (ii) because the natural histidine in maleimide (or Haloacetamide) group and albumen or the peptide, lysine, tyrosine or cysteine residues reaction; Crosslinked in the interchain of conjugates or the chain (A.Papini etc., Int.J.Pept.Protein Res., 1992; 39,348-355; T.Ueda etc., Biochemistry, 1985,24,6316-6322).This interchain linkage in the antibody will cause between heavy chain and the light chain or two heavy chains between various unreducible covalently bound, this is significantly in reduction SDS-PAGE analyzes, because exist than expection heavy chain and the more high-molecular weight band of light chain band.Crosslinkedly in this interchain or the chain in the antibody also can show, be different from the anomalous mass peak at prospective quality peak that antibody adds reporter group or the effect group of connection because exist through MS.Be different from traditional yoke and close method, the method for describing among the application causes having high homogeneity and the conjugates that do not have obvious interchain linkage or hydrolysis connector.
The clear and definite integral body by reference of all lists of references of quoting in this paper and following examples is incorporated into.
Embodiment
Only exemplary following examples are not to limit the present invention.
The traditional two-stage process of embodiment 1. contrasts uses Heterobifunctional connector maleimide-PEG through this method n-NHS yoke closes antibody and cytotoxic agent DM1/DM4 (Fig. 1).
Preparation concentration is the DM1 [N of 30-60mM in DMAC N,N (DMA) 2 '-deacetylation-N 2 '-(3-sulfydryl-1-oxopropyl)-maytansine] or DM4 [N 2 '-deacetylation-N 2 '-(4-sulfydryl-4-methyl isophthalic acid-oxo amyl group) maytansine] (DMx) mercaptan and maleimide-PEG nThe liquid storage of-NHS difunctionality connector.Connector and DMx mercaptan are containing 50%v/v 200mM succinate buffer at the most, and 2mM EDTA mixes among the DMA of pH 5.0 and obtains 1.6: 1 DMx: connector mol ratio and the DMx final concentration that equals 15mM.After the mixing; Make reaction mixture sat 1-4h; The aliquot reactant mixture is diluted 10 times and under 302-320nm, measure absorbance to confirm existing of any remaining unreacted maleimide then, uses the extinction coefficient (ε) of maleimide, 302nm=620M -1Cm -1With ε 320nm～450M -1Cm -1(afterwards the refrigerated reactant mixture of aliquot was carried out extra reversed-phase HPLC analysis and monitored absorbance to confirm when reactant mixture is added into antibody the formation of the connector of complete obiteration of connector maleimide and expectation-DMx reagent at 302nm and 252nm).When confirming no longer to have maleimide through UV, the aliquot reactant mixture need not purification and is added in phosphate buffer (pH 7.5) solution of antibody, the final yoke condition of closing is 4mg/ml Ab, 90% phosphate buffer/10%DMA, pH 7.5.Allow yoke to close reaction and at room temperature carry out 2h.Use is equilibrated G25 solvent resistant column or use tangential flow filtration (TFF) in pH 7.5 phosphate buffers, makes the Ab-DMx conjugates from excessive micromolecule DMx and connector reactant purification.Conjugates in pH 7.5 buffer 4 ℃ kept 2 days down further, to allow non-covalent connection or to dissociate through any DMx thing class that labile bond is connected to antibody.Then the conjugates dialysed overnight is got into pH 5.5 histidine/glycine buffer, filter with final storage through 0.22 μ m filter then.Measure the DMx number (on average) of in final conjugates each Ab molecule 252 with 280nm absorbance and use down known extinction coefficient of DMx and antibody under these two wavelength through measuring conjugates.
Use several kinds of different reaction conditions to be used for DMx mercaptan and Heterobifunctional maleimide-PEG 4The primary response of-NHS reagent: 50%DMA/50% aqueous 200mM succinate pH of buffer 5.0,2mM EDTA (v/v); Or 60%DMA/40%200mM succinate pH of buffer 5.0,2mM EDTA (v/v); Or 100%DMA, the organic base of every mole of DM4 mercaptan 1.5 molar equivalents (for example, N, N '-diisopropylethylamine, DIPEA or 4-methyl morpholine).
In a series of experiments, DMx and maleimide-PEG 4The molar equivalent of-NHS connector (available from Pierce Endogen) is 1.2-2.4, and the response time is 30min.The DMx/Ab number that the purification conjugates is measured is measured as the function that increases normal DMx/ antibody.1.2-2.0 the condition of equivalent DM1/ connector produces the conjugates with similar DMx/Ab carrying capacity, shows that the reaction that DMx mercaptan is not expected in the NHS of connector ester side is not a major issue.The crosslinked amount that exists in the final conjugates is also analyzed through reduction SDS PAGE, shows that the existence of crosslinked pollutant significantly reduces along with the DM1/ connector ratio that increases.
(before reactant mixture is added into antibody) introduced an optional quencher step using maleimide or Haloacetamide reagent (for example maleimide butanoic acid or maleimide propanoic acid or N-ethyl maleimide or iodoacetamide or iodoacetic acid) after initial DMx and Heterobifunctional connector reaction completion; With the excessive DMx mercapto of quencher, to prevent any reaction of not expecting of DMx mercaptan and antibody.
Antibody is included in initial reaction mixture and the antibody (when the reaction of DMx-connector is accomplished) that low pH (pH～5) mixes DMx and Heterobifunctional connector down with the optional method of the initial reaction mixture reaction of unpurified DMx and Heterobifunctional connector, adds buffer or alkali subsequently and closes reaction to increase pH to 6.5-8.5 to carry out yoke.
Antibody-PEG 4-Mal-DM1 or DM4 conjugates close the method preparation through two step of tradition yoke, compare with the yoke method of closing that the present invention describes.Concentration is that the humanized antibody of 8mg/ml is with excessive Heterobifunctional maleimide-PEG in pH 7.5 phosphate buffers (50mM potassium phosphate, 50mM sodium chloride, 2mM EDTA, pH 7.5) and 5%DMA 4-NHS connector reagent (available from Pierce Endogen) is modified.Behind 25 ℃ of following 2h, the antibody of modification through G25 chromatograph gel-purified to remove the excessive unreacted connector that does not add.Recovery through the Ab of UV absorbance measurement purification under 280nm.The TPPA that the number of the maleimide base group that in the Ab that modifies, connects uses aliquot to modify; Through adding the mercaptan (for example 2 mercapto ethanol) of known quantity, said mercaptan with the amount that surpasses maleimide add with modified antibodies in the reaction of maleimide residue and use DTNB reagent analysis residue mercaptan through the Ellman algoscopy then (mercaptan TNB be at extinction coefficient=14150M of 412nm -1Cm -1Riddles, P.W. etc., Methods Enzymol., 1983,91,49-60; Singh, R., Bioconjugate Chem., 1994,5,348-351).It is to carry out under the 2.5mg/ml that the yoke of modifying Ab and DM1 or DM4 mercaptan closes with Ab concentration in the reactant mixture, and said reactant mixture is by 95% phosphate buffer pH 7.5 (2mM EDTA, pH 7.5 for 50mM potassium phosphate, 50mM sodium chloride) and 5%DMA composition.The maleimide of the last every mole of connection of Ab adds the excessive DM1 or the DM4 mercaptan of 1.7 molar equivalents.After 25 ℃ of reactions are spent the night, conjugates use 0.22 μ m filter aseptic filtration and through equilibrated G25 post in phosphate buffer pH 7.5 (2mM EDTA, pH 7.5 for 50mM potassium phosphate, 50mM sodium chloride) from excessive unreacted DM1 or DM4 gel-purified.The conjugates of purification in phosphate buffer pH 7.5 (2mM EDTA, pH 7.5 for 50mM potassium phosphate, 50mM sodium chloride) 4 ℃ keep 2 days to allow non-covalent connection or to dissociate through any DM1 or DM4 thing class that labile bond is connected to antibody.In histidine/glycine buffer pH 5.5 (130mM glycine/10mM histidine, pH 5.5), conjugates was dialysed 2 days subsequently, and use 0.22 μ m filter aseptic filtration.Measure DM1 or the DM4 molecule number of in final conjugates each Ab molecule 252 with 280nm absorbance and use down known extinction coefficient of DM1/DM4 and Ab under these two wavelength through measuring conjugates.
The NuPage electrophoresis system (Invitrogen) that use has a 4-12%Bis Tris Gel reduces SDS PAGE to conjugates and antibody sample.Thermal denaturation with go back raw sample and load with 10 μ g/ swimming lanes.Use the reduction SDS-PAGE of the conjugates of the method preparation that the present invention describes only to show (Fig. 2) as the heavy chain of the expection of main band and light chain band (being respectively 50kDa and 25kDa).On the contrary, the conjugates that closes methods preparations through two step of tradition yokes shows that molecular weight is 75,100,125 and the crosslinked band of not expecting of 150kDa, infers to correspond respectively to interchain linkage thing class HL, H 2, H 2L and H 2L 2(Fig. 2).
Antibody-PEG that the method for describing through the present invention prepares 4Albumen LabChip electrophoretic analysis (under the reducing condition) show percent of-Mal-DM4 conjugates is the expection heavy chain and the light chain band of 58% and 30% (total protein), is similar to 65% and 30% (Fig. 3) that yoke not closes antibody respectively.On the contrary, the conjugates that uses two step of tradition yoke to close the method preparation shows respectively only 16% and 8% heavy chain and light chain band, and the main band of the higher molecular weight of scope 94-169kDa infers it is because interchain linkage.Analyze based on quantitative albumen LabChip, the conjugates height that the method for describing through the application prepares is superior to using the conjugates (Fig. 3) of traditional two-stage process preparation.
The MS of the conjugates that the method for describing through the present invention prepares analyzes and shows that relevant each antibody molecule has the isolating DMx-antibody conjugates peak (Fig. 4) that increases the maytansinoid of number molecule.On the contrary, use the MS of the conjugates that traditional two-stage process prepares almost to differentiate, show the inhomogeneity of conjugates goods, supposition is because the maleimide connector of crosslinked or inactivation.Therefore, based on MS, the conjugates that the method for using the present invention to describe prepares is superior to through the synthetic conjugates of traditional two-stage process.
The anti-CanAg Ab-PEG that the method for describing through the present invention prepares 4The combination of-Mal-DM1 conjugates is measured through flow cytometry, uses the COLO205 cell of antigen expressed, and finds to be similar to the combination that yoke not closes antibody, shows that yoke closes antagonist and combines not have adverse effect (Fig. 5).The anti-CanAg Ab-PEG that the method for describing through the present invention prepares 4The cellular cytoxicity activity of-Mal-DM1 conjugates uses expresses the antigenic COLO205 colon cancer cell of CanAg at in-vitro measurements (Fig. 6).The cancerous cell of antigen expressed is coated the cell culture medium that contains hyclone of 96 orifice plates with about 1000 cells/well and is exposed to the Ab-DMx conjugates of various concentration.Be exposed to conjugates after 5 days, the living cells that is retained in each hole uses WST-8 algoscopy (Dojindo Molecular Technologies) to measure.Anti-CanAg Ab-PEG as shown in Figure 6, as to use this method to prepare 4-Mal-DM1 conjugates is effective to expressing the antigenic COLO205 colon cancer cell of CanAg height under low concentration.The anti-CanAg Ab-PEG that the method for describing through the present invention prepares 4The cytotoxicity specificity of-Mal-DM1 conjugates is to the COLO205 cell, because this can close antibody and check through adding excessive not yoke.
Embodiment 2. contrast tradition are two-stage process continuously, uses maleimide-sulfo group-NHS connector yoke to close antibody and DM1/DM4 (Fig. 7) through this method.
Preparation concentration is the DMx mercaptan of 30-60mM and the liquid storage of maleimide-sulfo group-NHS Heterobifunctional connector in DMAC N,N (DMA).Connector and DMx mercaptan are containing 40%v/v 200mM succinate buffer, and 2mM EDTA mixes among the DMA of pH 5.0 and obtains 1.6 DMx: connector ratio and the DMx final concentration that equals 15mM.After the mixing, make reaction leave standstill 1-4h, the aliquot reactant mixture is diluted 10 times and under 302-320nm, measure absorbance existing with assessment reaction completion and maleimide then.(afterwards the refrigerated reactant mixture of aliquot was carried out extra reversed-phase HPLC analysis and monitored absorbance to confirm when reactant mixture is added into antibody the formation of the connector of complete obiteration of connector maleimide and expectation-DMx reagent at 302nm and 252nm).When confirming no longer to have maleimide through UV, the aliquot reactant mixture is added into the mixture of antibody in phosphate buffer (pH 7.5), the final yoke condition of closing is 4mg/ml Ab, 90% phosphate buffer/10%DMA, pH 7.5.Allow yoke to close reaction and at room temperature carry out 2h.Use equilibrated G25 solvent resistant column or through tangential flow filtration in pH 7.5 phosphate buffers makes the Ab-DMx conjugates from excessive unreacted DMx and the connector purifying products that closes of yoke not.Conjugates in pH 7.5 buffer 4 ℃ kept 2 days down, to allow non-covalent connection or to dissociate through any DMx thing class that labile bond is connected to antibody.Then the conjugates dialysed overnight is got into pH 5.5 histidine/glycine buffer, filter with final storage through 0.22 μ m filter then.Measure the DMx molecule number (on average) of in final conjugates each Ab molecule 252 with 280nm absorbance and use down known extinction coefficient of DMx and antibody under these two wavelength through measuring conjugates.
For relatively, use the conventional two step yoke methods of closing to prepare Ab-sulfo group-Mal-DMx conjugates.Concentration is that the antibody (Ab) of 8mg/ml is modified with excessive Heterobifunctional maleimide sulfo group-NHS connector in pH 7.5 phosphate buffers/5%DMA buffer.Allow to be reflected at and carry out 2h under 20 ℃, the Ab that modifies then uses G25 chromatograph and excessive unreacted connector purifies and separates.Recovery through the Ab of UV absorbance measurement purification under 280nm.The TPPA that the number of the maleimide base group that in the Ab that modifies, connects uses aliquot to modify; Through adding the mercaptan (for example 2 mercapto ethanol) of known quantity, said mercaptan with the amount that surpasses maleimide add with modified antibodies in the reaction of maleimide residue and use DTNB reagent analysis residue mercaptan through the Ellman algoscopy then (mercaptan TNB be at extinction coefficient=14150M of 412nm -1Cm -1Riddles, P.W. etc., Methods Enzymol., 1983,91,49-60; Singh, R., Bioconjugate Chem., 1994,5,348-351).It is to carry out under the 2.5mg/ml that Ab that modifies and the yoke of DMx close with AC among 95%pH 7.5 phosphate buffers/5%DMA (v/v), and the maleimide of every mole of connection adds the DMx mercaptan of 1.7 molar equivalents in the antibody.Be reflected at 18 ℃ and carry out 8-24h, conjugates separates with excessive unreacted DMx through using the G25 size exclusion chromatography.Behind the purification, conjugates in pH 7.5 buffer 4 ℃ keep 2 days to allow non-covalent connection or to dissociate through any DMx thing class that labile bond is connected to antibody.Conjugates is got into pH 5.5 histidine/glycine buffer by dialysed overnight then, filters with final storage through 0.22 μ m filter then.Measure the DMx molecule number of in final conjugates each Ab molecule 252 with 280nm absorbance and use down known extinction coefficient of DMx and antibody under these two wavelength through measuring conjugates.
Use has the NuPage electrophoresis system (Invitrogen) of 4-12%Bis Tris Gel and NuPAGE MOPS SDS electrophoretic buffer, uses conjugates and antibody sample (10 μ g/ swimming lane) to reduce SDS PAGE (Fig. 8).On the gel molecular weight be 75,125 and the band of 150kDa indication interchain linkage thing class (be respectively HL, H 2L and H 2L 2).The high molecular weight crosslinked thing class with much little ratios is compared in the more clearly demonstration of Ab-sulfo group-Mal-DM1 conjugates and～4DM1/Ab (be respectively through the swimming lane 3 of this method with through two step of tradition yokes and close the swimming lane 2 of method) and～6DM1/Ab (be respectively through the swimming lane 5 of this method with through two step of tradition yokes and close the swimming lane 4 of method), the conjugates (swimming lane 3 and 5) of the method preparation of describing through the present invention with the conjugates (swimming lane 2 and 4) for preparing through traditional two-stage process.
Albumen LabChip electrophoretic analysis (under the reducing condition) show percent of the antibody-sulfo group-Mal-DM1 conjugates of the method preparation of describing through the present invention is the heavy chain and the main band of light chain of 70% and 28% (total protein), is similar to 70% and 30% (Fig. 9) that yoke not closes antibody respectively.On the contrary, use the conjugates of traditional two-stage process preparation to show respectively only 53% and 23% heavy chain and light chain band, and the main band of the higher molecular weight of scope 99-152kDa infer it is because interchain linkage.Analyze based on quantitative albumen LabChip, the conjugates of the method preparation of describing through the application is lacking the conjugates (Fig. 9) that height aspect the interchain linkage is superior to using traditional two-stage process preparation.
Analyze relatively through the present invention method of describing and the Ab-sulfo group with the similar drug load-mal-DM1 conjugates (Figure 10) for preparing through traditional two-stage process through size exclusion LC/MS.The conjugates of the method preparation of describing through the present invention shows that only containing quality equals Ab-(connector-DMx) nThe expection MS spectrum that distributes of expection peak.Under the situation of the conjugates that uses traditional two-stage process preparation, main peaks is except the Ab-of expection (connector-DMx) in the spectrum nPart all contains one or more hydrolysis or crosslinked connector fragment.Interchain supposition mechanism crosslinked or maleimide aqueous inactivation is shown in Figure 17 in the tradition two-step reaction order; Thus maleimide (or Haloacetamide) residue that adds of the primary response through antibody and Heterobifunctional connector can with intramolecularly (or intermolecular) histidine, lysine, tyrosine or cysteine residues reaction; Cause interchain linkage, perhaps initial maleimide (or Haloacetamide) residue that adds can be by inactivation (for example separate through hydrolysis maleimide ring crack or be added into maleimide through aqueous) and therefore for unavailable with the fast reaction of effect group that contains mercaptan or reporter group.Therefore, LC-MS analyzes clear the demonstration, and the method that the present invention describes helps producing the homogeneous conjugates, has seldom or the hydrolysis that is not connected with antibody or crosslinked connector fragment.
The COLO205 cell that is used in combination antigen expressed of anti-CanAg Ab-sulfo group-Mal-DM1 (5.6 maytansinoids of each antibody molecule load (on average)) that the method for describing through the present invention prepares is measured through flow cytometry; And find to be similar to the combination that yoke not closes antibody, show combine (Figure 11) that yoke closes does not influence antibody and target antigen.The cellular cytoxicity activity of anti-CanAg Ab-sulfo group-Mal-DM1 conjugates that the method for describing through the present invention prepares uses expresses the antigenic COLO205 colon cancer cell of CanAg at in-vitro measurements (Figure 12).The cancerous cell of antigen expressed is coated the cell culture medium that contains hyclone of 96 orifice plates with about 1000 cells/well and is exposed to the Ab-DMx conjugates of various concentration.Be exposed to conjugates after 5 days, the living cells of reservation uses WST-8 algoscopy (Dojindo Molecular Technologies) to measure.Shown in figure 12, use the anti-CanAg Ab-sulfo group-Mal-DM1 conjugates of this method preparation effective to expressing the antigenic COLO205 colon cancer cell of CanAg height under low concentration.The cytotoxicity of this conjugates is specific, because the competition of this antibody that can close with excessive not yoke is checked.
The optional method that uses describing method yoke of the present invention to close be included in the reaction of initial DMx and Heterobifunctional connector accomplish after (before reactant mixture is added into antibody) use maleimide or Haloacetamide reagent (for example 4-maleimide butanoic acid or 3-maleimide propanoic acid or N-ethyl maleimide or iodoacetamide or iodoacetic acid) the quencher step with the excessive DMx mercapto of quencher, to prevent any reaction of not expecting of DMx mercaptan and antibody.In specific embodiment; After initial DMx and Heterobifunctional connector reaction completion (before reactant mixture is added into antibody); Add 4-maleimide butanoic acid with the excessive DMx mercapto of quencher, close any reaction of not expecting of DMx mercaptan and antibody between the reaction period to prevent yoke.Reactant mixture to the DM4 that contains excessive DM4 (3mM) and sulfo group-Mal-NHS Heterobifunctional reagent; When the maleimide base group that is coupled to Heterobifunctional reagent at the DM4 mercaptan of expection is accomplished, the 4-maleimide butanoic acid (6mM) that at room temperature adds the twice molar excess to reactant mixture continue 20 minutes with quencher from the remaining DM4 of initial coupling reaction.Need not the purification reaction mixture, aliquot mixes with the solution of the phosphate buffer (pH 7.5) of antibody, and the final yoke condition of closing is 4mg/ml Ab, 90% aqueous phosphate buffer/10%DMA, and pH 7.5.Allow yoke to close reaction and at room temperature carry out 2h.Use equilibrated G25 solvent resistant column in pH 7.5 phosphate buffers makes antibody-DM4 conjugates from excessive micromolecule DM4 and connector reactant purification.Conjugates in pH 7.5 buffer 4 ℃ kept 2 days down further, to allow non-covalent connection or to dissociate through any DMx thing class that labile bond is connected to antibody.Then the conjugates dialysed overnight is got into pH 5.5 histidine/glycine buffer, and filter with final storage through 0.22 μ m filter.Measure the DM4 molecule average number of in final conjugates each Ab molecule 252 with 280nm absorbance and use down known extinction coefficient of DMx and antibody under these two wavelength through measuring conjugates.Use has the NuPage electrophoresis system (Invitrogen) of 4-12%Bis Tris Gel, analyzes the conjugates sample through non-reduced SDS PAGE.The thermal denaturation sample loads with 10 μ g/ swimming lanes.Use the non-reduced SDS-PAGE of the conjugates (not quencher) of the method preparation that the present invention describes show the light chain band (～25kDa) with incomplete antibody band (heavy chain-light chain;～75kDa) evidence (Figure 18).On the other hand, use conjugates method preparation that the present invention describes, that handle (to add the excessive DMx mercaptan of medicated cap) with 4-maleimide butanoic acid to have significantly lower these bands of not expecting of measuring (with the level of the antibody sample that is equivalent to unmodified).Through the mercaptan quencher for example the initial DMx of 4-maleimide butanoic acid quencher and Heterobifunctional reactant mixture (and before the antibody yoke closes) another advantage be to close between the reaction period and do not have " free " DMx (DM1 or DM4) thing class at the antibody yoke, and therefore the final conjugates behind the purification does not contain " free " or the yoke DMx thing class of closing not.The adduct of DMx and 4-maleimide butanoic acid (or other polarity mercaptan quencher reagent) is more water-soluble than DMx, and therefore can separate with covalently bound antibody-DMx conjugates more easily.
Embodiment 3. uses sulfo group-NHS-SMCC connector yokes to close antibody and maytansinoid (DM1/DM4) (Figure 13)
Preparation concentration is that DM1 or the DM4 mercaptan (DMx) of 30-60mM and the sulfo group-SMCC Heterobifunctional connector with sulfo group-NHS group are (available from Pierce Endogen in DMA; Liquid storage Figure 13).Connector and DM1 or DMx mercaptan are containing 40%v/v 200mM aqueous succinate buffer, and 2mM EDTA mixes among the DMA of pH 5.0 and obtains 1.6: 1 DM1 or DM4 (DMx): the DMx final concentration of connector ratio and 6mM.After the mixing, make to be reflected at room temperature and to leave standstill 1-4h, then the aliquot reactant mixture diluted 10 times under 302-320nm, to measure absorbance whether all maleimides react with assessment.(afterwards the refrigerated reactant mixture of aliquot was carried out extra reversed-phase HPLC analysis and monitored with confirmation when reactant mixture is added into antibody the formation of the sulfo group of complete obiteration of connector maleimide and expectation-NHS-connector-Mal-DMx reagent at 302nm and 252nm).When confirming no longer to have maleimide through UV, the aliquot reaction is added into the aqueous solution of antibody in phosphate buffer (pH 7.5), the final yoke condition of closing is 4mg/ml Ab, 90% phosphate buffer (aqueous)/10%DMA (v/v), pH 7.5.Allow yoke to close reaction and at room temperature carry out 2h.Use is equilibrated G25 solvent resistant column in pH 7.5 phosphate buffers (aqueous), makes the Ab-DMx conjugates from excessive unreacted reagent and excessive DMx purification.Conjugates in pH 7.5 buffer 4 ℃ kept 2 days down, to allow non-covalent connection or to dissociate through any DMx thing class that labile bond is connected to Ab.Then the conjugates dialysed overnight is got into pH 5.5 histidine/glycine buffer, filter with final storage through 0.22 μ m filter then.Measure the DMx molecule number of in final conjugates each Ab molecule 252 with 280nm absorbance and use down known extinction coefficient of DMx and antibody under these two wavelength through measuring conjugates.
For relatively, use the conventional two step yoke methods of closing to prepare the Ab-SMCC-DMx conjugates.Concentration is that the antibody (Ab) of 8mg/ml is modified with the excessive sulfo group with sulfo group-NHS group-SMCC connector (available from Pierce Endogen) in 95%pH 6.5 phosphate buffers/5%DMA buffer.Allow to be reflected at and carry out 2h under 25 ℃, the Ab that modifies then uses G25 chromatograph and excessive unreacted connector purifies and separates.Recovery through the Ab of UV absorbance measurement purification under 280nm.The TPPA that the number of the maleimide base group that in the Ab that modifies, connects uses aliquot to modify; Through adding the mercaptan (for example 2 mercapto ethanol) of known quantity, said mercaptan with the amount that surpasses maleimide add with modified antibodies in the reaction of maleimide residue and use DTNB reagent analysis residue mercaptan through the Ellman algoscopy then (mercaptan TNB be at extinction coefficient=14150M of 412nm -1Cm -1Riddles, P.W. etc., Methods Enzymol., 1983,91,49-60; Singh, R., Bioconjugate Chem., 1994,5,348-351).It is to carry out under the 2.5mg/ml that Ab that modifies and the yoke of DM1 or DM4 close with AC in 95%pH6.5 phosphate buffer/5%DMA (v/v), and the maleimide of every mole of connection adds the DM1 or the DM4 mercaptan of 1.7 molar equivalents among the Ab.Be reflected at 18 ℃ and carry out 8-24h, conjugates separates with excessive unreacted DM1 (or DM4) through the G25 chromatograph.Behind the purification, conjugates in pH 6.5 buffer 4 ℃ keep 2 days to allow any weak DM1/DM4 thing class hydrolysis that connects.Conjugates is got into pH 5.5 histidine/glycine buffer by dialysed overnight then, filters with final storage through 0.22 μ m filter then.Measure the DM1/DM4 molecule number of in final conjugates each Ab molecule 252 with 280nm absorbance and use down known extinction coefficient of DM1/DM4 and antibody under these two wavelength through measuring conjugates.
The NuPage electrophoresis system (Invitrogen) that use has 4-12%Bis Tris Mini Gel and a NuPAGE MOPS SDS electrophoretic buffer reduces SDS PAGE (Figure 14) to conjugates and antibody sample (10 μ g/ swimming lane).On the gel molecular weight be 75,125 and the band of 150kDa indication interchain linkage thing class (be respectively HL, H 2L and H 2L 2).The more clearly demonstration of Ab-SMCC-DM1 conjugates and 3.1D/Ab (being respectively swimming lane 4 and the swimming lane 3 through traditional two-stage process through this method), the conjugates (swimming lane 4) of the method preparation of describing through the present invention is compared the high molecular weight crosslinked thing class with much less with the conjugates (swimming lane 3) for preparing through traditional two-stage process.
Albumen LabChip electrophoretic analysis (under the reducing condition) show percent of the antibody-SMCC-DM1 conjugates of the method preparation of describing through the present invention is the heavy chain and the main band of light chain of 67% and 30% (total protein), is similar to 68% and 30% (Figure 15) that yoke not closes antibody respectively.On the contrary, use the conjugates of traditional two-stage process preparation to show respectively only 54% and 24% heavy chain and light chain band, and the main band of the higher molecular weight of scope 96-148kDa infer it is because interchain linkage.Analyze based on quantitative albumen LabChip, the conjugates of the method preparation of describing through the application is lacking the conjugates (Figure 15) that height aspect the interchain linkage is superior to using traditional two-stage process preparation.
Analyze relatively through the present invention method of describing and the Ab-SMCC-DM1 conjugates (Figure 16) for preparing through traditional two-stage process through size exclusion LC/MS with similar drug load.The conjugates of the method preparation of describing through the present invention shows that only containing quality equals Ab-(connector-DMx) nThe expection MS spectrum that distributes of expection peak.Under the situation of the conjugates that uses traditional two-stage process preparation, spectrum shows heterogeneous thing class mixture, comprises the Ab-(connector-DMx) of expection nThing class and maleimide and segmental other thing classes of crosslinked connector of containing inactivation.Interchain supposition mechanism crosslinked or the maleimide inactivation is shown in Figure 17 in the tradition two-step reaction order; Thus the maleimide (or Haloacetamide residue) that adds of the primary response through antibody and Heterobifunctional connector can with intramolecularly (or intermolecular) histidine, lysine, tyrosine or cysteine residues reaction; Cause interchain linkage, perhaps initial maleimide (or Haloacetamide) residue that adds can with DM1 that contains mercaptan or DM4 (DMx) substance reaction step before through hydrolysis or hydration maleimide residue and inactivation.Therefore, LC-MS analyzes clear the demonstration, and the method that the present invention describes helps producing the homogeneous conjugates, has seldom or the maleimide of the inactivation that is not connected with antibody or crosslinked connector fragment.
Embodiment 4. uses the disulphide connector yoke of cleavable to close antibody and DM1/DM4 (DMx) (Figure 19) through this method.
Preparation concentration is the DM1 of 30-60mM or the liquid storage of DM4 mercaptan (DMx) and Heterobifunctional connector 4-(2-pyridine radicals disulfide group) butanoic acid-N-hydroxy-succinamide ester (SPDB) in DMA.Connector and DMx mercaptan are containing nearly 40%v/v 200mM aqueous succinate buffer, and 2mM EDTA mixes among the DMA of pH 5.0 and obtains 1.6: 1 DM1 or DM4 (DMx): the DMx final concentration of connector ratio and 8mM.After the mixing, make to be reflected at room temperature and to leave standstill 1h, the aliquot reaction is added into the aqueous solution of antibody in phosphate buffer (pH 7.5) then, and the final yoke condition of closing is 4mg/ml Ab, 90% phosphate buffer (aqueous)/10%DMA, and pH 7.5.Allow yoke to close reaction and at room temperature carry out 2h.Use is equilibrated G25 solvent resistant column in pH 7.5 phosphate buffers (aqueous), makes the Ab-DMx conjugates from excessive unreacted reagent and excessive DMx purification.Conjugates in pH 7.5 buffer 4 ℃ kept 2 days down, to allow non-covalent connection or to dissociate through any DMx thing class that labile bond is connected to Ab.Then the conjugates dialysed overnight is got into pH 5.5 histidine/glycine buffer, filter with final storage through 0.22 μ m filter then.Measure the DMx molecule number of in final conjugates each Ab molecule 252 with 280nm absorbance and use down known extinction coefficient of DMx and antibody under these two wavelength through measuring conjugates.
Embodiment 5. through this method use the disulphide connector and not the cleavable connector prepare antibody-DM1/DM4 (Ab-DMx) conjugates (Figure 20).
Preparation concentration is DM1 or DM4 mercaptan (DMx) and the NHS-PEG of 30-80mM in DMAC N,N (DMA) nThe liquid storage of-maleimide Heterobifunctional connector.NHS-PEG 4-maleimide connector and DMx mercaptan are containing 40%v/v 200mM succinate buffer, and 2mM EDTA mixes among the DMA of pH 5.0 and obtains 1.6: 1 DMx: connector mol ratio and the DMx final concentration that equals 8.0mM.Make reactant mixture continue reaction 2h in room temperature.In independent parallel reaction, SPDB connector and DMx mercaptan mix and react to being used for NHS-PEG in a similar manner 4The condition of-maleimide reaction continues the response time of 1h.Purification not after reaction is accomplished merges isopyknic PEG 4-Mal-DM4 mixture and SPDB-DM4 mixture.The reactant mixture that unpurified aliquot is merged is added into the solution of antibody in phosphate buffer (pH 7.5), and the final yoke condition of closing is 4mg/ml Ab, 90% phosphate buffer (aqueous)/10%DMA (v/v), and pH 7.5.Allow yoke to close reaction and at room temperature carry out 2h.Use is equilibrated G25 solvent resistant column in pH 7.5 phosphate buffers (aqueous), makes the Ab-DMx conjugates from excessive unreacted reagent and excessive DMx purification.Conjugates in pH 7.5 buffer 4 ℃ kept 2 days down, to allow non-covalent connection or to dissociate through the DMx thing class that labile bond is connected to Ab.Then the conjugates dialysed overnight is got into pH 5.5 histidine/glycine buffer, filter with final storage through 0.22 μ m filter then.Measure the DMx molecule number of in final conjugates each Ab molecule 252 with 280nm absorbance and use down known extinction coefficient of DMx and antibody under these two wavelength through measuring conjugates.
Through DTT (dithiothreitol, DTT) relatively handle conjugates with before the reduction disulfide bond with afterwards DMx/ antibody (D/A) ratio, Ab-(blended SPDB and PEG that the method for describing through the present invention is prepared 4-Mal connector)-DMx conjugates test to be to measure among the Ab cleavable and the adding percentage ratio of cleavable connector not.For the pH that keeps between the DTT reduction period is 7.5, conjugates is at first got into 250mM HEPES pH of buffer 7.5 by dialysis.Reduce conjugates through down reacting 20min then with 25mM DTT at 37 ℃.After the DTT reaction, use equilibrated G25 solvent resistant column discharges from the reactant mixture separation in 250mM HEPES pH of buffer 7.5 DMx and DTT.Measure the DMx molecule average number of in purified product each Ab molecule 252 with 280nm absorbance and use down known extinction coefficient of DMx and antibody under these two wavelength through measuring conjugates.Ratio between the D/A of the D/A of the conjugates that DTT handles and the conjugates of non-DTT processing is used to calculate the percentage ratio that is connected to the DMx of Ab through cleavable key not.Two other samples Ab-SPDB-DM4 and Ab-PEG 4-Mal-DM4 conjugates is handled with DTT, respectively as positive control and negative control.Handle before and D/A ratio afterwards through comparing DTT, contrast can not cracked Ab-PEG 4-Mal-DM4 conjugates shows, finds that nearly all bonded connector such as expection are can not cracked (93%).Containing of the method preparation of describing through the present invention can not cracked connector and Ab-(the blended SPDB and the PEG of disulphide connector 4The Mal connector)-the DMx conjugates has less with respect to the DMx loss amount of the Ab-SPDB-DMx conjugates of being made up of the cleavable connector fully that 41% the DTT that passes through handles cracked DMx.This explanation, Ab-(blended SPDB and PEG that the method for describing through the present invention prepares 4-Mal)-the DMx conjugates can not cracked connector by about 40% and the connector of 60% cleavable constitute.Can not cracked connector reagent and the initial ratio of the connector reagent of cleavable through changing, antibody and the conjugates of maytansinoid or other effectors can use different ratios can not cracked connector and the connector of cleavable prepare.Figure 21 has shown the mass spectrum of above-mentioned desaccharide base conjugates, and it comprises having through disulphide connector (SPDB) and antibody that can not the two average 3.5 maytansinoid molecule of each antibody molecule that are connected of cracked connector (PEG).MS shows and to have the cleavable and the independent conjugates thing class (Figure 21) of cleavable connector not.For example, the conjugates peak that is called D2-PEG-SPDB has a maytansinoid molecule that a disulphide connects and a maytansinoid molecule that can not cracked thioether be connected; The conjugates peak that is called D3-PEG-2SPDB has a maytansinoid molecule that two disulphide connect and a maytansinoid molecule that can not cracked thioether be connected; The conjugates peak that is called D3-2PEG-SPDB has maytansinoid molecule that a disulphide connects and two maytansinoid molecules that can not cracked thioether be connected.
Embodiment 6. uses SMCC connector yoke to close antibody and maytansinoid (Figure 22).
Preparation concentration is the DM1 mercaptan of 30-60mM and the liquid storage of SMCC Heterobifunctional connector (Pierce) in DMA.Connector and DM1 mercaptan are containing nearly 50%v/v 200mM aqueous succinate buffer, and 2mM EDTA mixes among the DMA of pH 5.0 and obtains the DM1 of 1.4: 1 molar equivalents: the connector ratio with equal 1 to 6mM DM1 final concentration.After the mixing, make to be reflected at room temperature and to leave standstill 4h, then the aliquot reactant mixture diluted 10 times with under 302-320nm, measure absorbance with the whether all maleimide of assessment with thiol reactant.When confirming no longer to have maleimide through UV, the aliquot reaction is added into the aqueous solution of antibody in phosphate buffer (pH 7.5-8.5), the final yoke condition of closing is 2.5mg/ml Ab, 70-80% phosphate buffer (aqueous)/30-20%DMA (v/v).Allow yoke to close reaction and at room temperature carry out 3h.Use is equilibrated G25 solvent resistant column in pH 7.4 phosphate buffers (aqueous), makes reagent and the excessive DM1 purification of Ab-DM1 conjugates from excessive unreacted or hydrolysis.Then the conjugates dialysed overnight being got into pH 7.4 phosphate buffers (aqueous) also filters with final storage through 0.22 μ m filter then.Measure the DM1 molecule number of in final conjugates each Ab molecule 252 with 280nm absorbance and use down known extinction coefficient of DM1 and antibody under these two wavelength through measuring conjugates.The conjugates that can prepare similarly, antibody and DM4 mercaptan and SMCC.The antibody of use SMCC connector and these conjugatess of DM1 or DM4 contain thioether can not cracked connector.
The MS of the Ab-SMCC-DM1 conjugates of the method preparation of describing through the present invention through the de-glycosylation conjugates analyzes and identifies (Figure 23).The conjugates of the method preparation of describing through the present invention shows that containing quality equals Ab-(connector-DM1) nThe expection MS spectrum that distributes of expection peak.
Embodiment 7. uses the connector that contains Heterobifunctional disulphide, and (SSNPB, SPP) yoke closes antibody and maytansinoid.
Through being similar to the similar method of describing to SPDB connector among the embodiment 4 of method, the disulphide that contains Heterobifunctional connector SSNPB (N-thiosuccimide base-4-(5-nitro-2-pyridine radicals disulfide group) butyrate) and SPP (N-succinimido-3-(2-pyridine radicals disulfide group) propionic ester) can be used for preparing antibody-maytansinoid conjugates that disulphide is connected.The structure of the conjugates that the disulphide that uses SPDB (Figure 19) to prepare connects is identical with the structure of the conjugates for preparing with SSNPB (Figure 24).The MS display quality value of the conjugates that the disulphide that uses SPDB to prepare connects is corresponding to the discrete peak of the different numbers of the maytansinoid molecule that is connected to antibody.
Embodiment 8. antibody with contain have the linear alkyl carbochain can not cracked connector the yoke of maytansinoid close.
Be similar to the method for describing to the SMCC connector among the embodiment 6, use the reactant mixture of maytansinoid and Heterobifunctional connector to prepare the conjugates that contains not cleavable connector with linear alkyl carbochain with linear alkyl carbochain.For example, shown in figure 26, the conjugates use BMPS of humanized antibody and DM1 (N-[β-dimaleoyl imino propoxyl group succinimide ester) or GMBS ((N-[γ-dimaleoyl imino butoxy] succinimide ester) connector preparation.At 60%DMA/40% (v/v) 200mM succinate buffer, the initial reaction mixture that contains BMPS or GMBS (8mM) and DM1 mercaptan (10.4mM) among the pH 5 shows when maleimide amine moiety complete reaction (being based on the decline of maleimide absorbance under the 302-320nm) when detecting in 15 minutes.Divided two parts that reactant mixture is added into the 80%EPPS aqueous buffer solution at interval with 30 minutes, the humanized antibody solution of 2.5mg/ml among the pH 8.1, this solution contains 20%DMA (v/v), and total connector is to add with antibody 8 molar equivalents.Conjugates mixture gel filtration and stand 2 and take turns dialysis behind 4h.DM1/ antibody ratio is that 3.8 and 5.1 conjugates reclaims with high monomer % (96.2-97.6%) with 71-75% and prepares.Analyze through HISEP HPLC with these conjugatess of GMBS or BMPS preparation and to show the free drug that does not have yoke not to close.Shown in figure 25; The similar conjugates that contains the not cleavable connector with linear alkyl chain can use AMAS (N-[β-dimaleoyl imino acetoxyl group] succinimide ester) or EMCS (N-[β-dimaleoyl imino hexylyloxy] succinimide ester) or sulfo group-N-hydroxy-succinamide ester (sulfo group-GMBS, sulfo group-EMCS) preparation.Table 1 has shown through the monomer % of the selected conjugates of describing method preparation of the present invention, has all shown high monomer % through the size exclusion chromatography analysis.For relatively, also shown the monomer % that closes the conjugates of method (through antibody and Heterobifunctional connector primary response, subsequently with the CHROMATOGRAPHIC FRACTIONATION AND MASS thiol reactant) preparation through two step of tradition yoke.
Table 1. is through the application method of describing and the monomer % that closes the selected conjugates of method preparation through two step of tradition yoke.
Conjugates D/A Yoke closes method The % monomer
Ab-PEG 4-Mal-DM1 6.6 The present invention 99.0
Ab-PEG 4-Mal-DM1 6.8 Two steps 98.0
Ab-sulfo group-Mal-DM1 3.6 The present invention 99.0
Ab-sulfo group-Mal-DM1 4.0 Two steps 96.7
Ab-SMCC-DM1 4.0 The present invention 98.6
Ab-SMCC-DM1 3.8 Two steps 97.0
Ab-PEG 4-Mal-DM4 6.2 The present invention 96.9
Ab-PEG 4-Mal-DM4 6.1 Two steps 84.5
Ab-SPDB-DM4 4.1 The present invention 99.4
Ab-SPDB-DM4 3.9 Two steps, one jar 95.7

Claims (17)

1. the method for conjugates of a preparation purification in solution; Wherein said conjugates comprises effector molecule or the reporter molecule that is connected with the cell node mixture; Said method comprising the steps of: (a) make said effector molecule or reporter molecule contact with difunctionality connector reagent with covalently bound said connector and said effector molecule or reporter molecule and thus preparation comprise the said effector molecule with the connector that is attached thereto or unpurified first mixture of reporter molecule; (b) through the reaction of said unpurified first mixture and cell node mixture said cell node mixture and said effector molecule with the connector that is attached thereto or reporter molecule yoke are closed to prepare second mixture and (c) to make said second mixture stand tangential flow filtration, dialysis, gel filtration, adsorption chromatography, selective precipitation or its combination to prepare the conjugates of purification thus.
2. the described method of claim 1, wherein step (b) is carried out to about 9 solution at pH about 4.
3. the described method of claim 1, wherein second mixture of step (b) is gone up basically and is not contained the thing class of not expecting crosslinked, hydrolysis that generates owing to intramolecularly or intermolecular reaction.
4. the described method of claim 1, wherein said effector molecule is a cytotoxic agent.
5. the described method of claim 4, wherein said cytotoxic agent is maytansinoid, taxane, CC1065 or its analog.
6. the described method of claim 4, wherein said cytotoxic agent is a maytansinoid.
7. the described method of claim 6, wherein said maytansinoid comprises mercapto.
8. the described method of claim 6, wherein said maytansinoid is DM1.
9. the described method of claim 6, wherein said maytansinoid is DM4.
10. the described method of claim 1, wherein said cell node mixture is interferon, interleukin-22 (IL-2), interleukin-13 (IL-3), interleukin 4 (IL-4), interleukin 6 (IL-6), insulin, EGF, TGF-α, FGF, G-CSF, VEGF, MCSF, GM-CSF, transferrin or antibody.
11. the described method of claim 10, wherein said cell node mixture is an antibody.
12. the described method of claim 11, wherein said antibody is monoclonal antibody.
13. the described method of claim 11, wherein said antibody are people or Humanized monoclonal antibodies.
14. the described method of claim 10, wherein said antibody are MY9, anti-B4, EpCAM, CD2, CD3, CD4, CD5, CD6, CD11, CD19, CD20, CD22, CD26, CD30, CD33, CD37, CD38, CD40, CD44, CD56, CD79, CD105, CD138, EphA receptor, EphB receptor, EGFR, EGFRvIII, HER2, HER3, mesothelium element, cripto, α vβ 3, α vβ 5, α vβ 6Integrin or C242.
15. the described method of claim 13, wherein said people or humanized antibody be My9-6, B4, C242, N901, DS6, EphA2 receptor, CD38, IGF-IR, CNTO 95, B-B4, Qu Sizhu monoclonal antibody, the appropriate strain monoclonal antibody of handkerchief, than cutting down pearl monoclonal antibody, sibrotuzumab or Rituximab.
16. the described method of claim 1, wherein said connector are cleavables or can not cracked connector.
17. the described method of claim 1, wherein said reporter molecule is a radiosiotope.
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